Generation of synthetic genomes

ABSTRACT

Methods for generating synthetic genomes, for example synthetic genomes having desired properties or viable genomes of reduced size, are disclosed. Also disclosed are synthetic genomes produced by the methods disclosed herein and synthetic cells containing the synthetic genomes disclosed herein.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62/312,398, filed on Mar. 23, 2016, entitled “GENERATION OF SYNTHETIC GENOMES,” the content of which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED R&D

This invention was made with government support under Contract Nos. HR0011-12-C-0063 and HR0011-16-2-0010 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.

BACKGROUND Field

The present application relates generally to molecular biology, and more particularly to synthetic genomes.

Description of the Related Art

Methods and techniques for producing and modifying cellular genomes are useful in the field of cell biology, in particular deciphering the operating system of the cell. Genome reductions of bacterial cells have been achieved by a series of sequential deletion events to facilitate the goal of understanding the molecular and biological function of genes essential for life. After each deletion, viability, growth rate, and other phenotypes of the resulting bacterial genome with a reduced size were determined. However, there is still a need for more systematic and improved method for designing and producing synthetic genomes of interest, and to improve our ability to identify essential genes.

SUMMARY

A method for generating a synthetic genome of interest is disclosed herein. In some embodiments, the method comprises: (a) providing a first genome; (b) designing a second genome based on the first genome, wherein the second genome is hypothesized to have a set of desired properties; (c) dividing each of the first and second genomes into N corresponding fragments, wherein N is an integer equal to or greater than 3; (d) combining at least one fragment of the second genome with fragments of the first genome to generate a third genome having all N corresponding fragments; (e) testing the third genome generated in step (d) for the set of desired properties; and (f) identifying the third genome as a synthetic genome of interest if it has the set of desired properties; otherwise modifying at least one fragment of the second genome and repeating steps (d)-(f) in one or more iterations until a set of desired properties is obtained in the third genome. The first genome can be, for example, a naturally occurring genome. In some embodiments, the first genome is a genome of unicellular organism. In some embodiments, the first genome is a bacterial genome, a yeast genome, a single-cell alga genome, or a combination thereof. In some embodiments, the first genome is a single chromosome genome. In some embodiments, the first genome is a multi-chromosome genome.

In some embodiments, step (b) comprises testing the second genome for the set of desired properties. In some embodiments, designing the second genome comprises modifying the first genome based on the information from literature resources, experimental data, or any combination thereof. In some embodiments, the experimental data comprises data obtained from a mutation study of the first genome, a genome related to the first genome, or any combination thereof. In some embodiments, the experimental data comprises data related to genes of essential function redundancies (EFR). The mutation study can comprise, for example, one or more of mutagenesis study, gene knockout study, and add-back study. The mutagenesis study can comprise, for example, random mutagenesis, targeted mutagenesis, or both. The mutageneis study comprises, in some embodiments, transposon-based mutagenesis, insertional mutagenesis, or both.

In some embodiments, all of the N corresponding fragments are substantially the same length. In some embodiments, at least two of the N corresponding fragments are different in length. In some embodiments, at least one of the N corresponding fragments is a chromosome of the first or second genome. In some embodiments, at least one of the N corresponding fragments is a portion of a chromosome of the first or second genome.

In some embodiments, testing the genome for the set of desired properties comprises introducing the genome into a cell or a cell-like system. The genome can be introduced into the cell or the cell-like system through, for example, conjugation, transformation, transduction, or any combination thereof. In some embodiments, the cell-like systems comprises a membrane-bound volume, a lipid vesicle, a cell from which one or more intracellular components have been removed, a cell from which the resident genome has been removed, or any combination thereof.

In some embodiments, modifying at least one of the second genome fragments in step (f) is at least partly based on the testing of step (e). In some embodiments, modifying at least one fragment of the second genome in step (f) further comprises conducting mutation study of the at least one fragment and modifying the at least one fragment at least partly based on the mutation study. In some embodiments, the mutation study comprises one or more of mutagenesis study, gene knockout study, and add-back study. The mutagenesis study can comprise, for example, random mutagenesis, targeted mutagenesis, or both. The mutageneis study comprises, in some embodiments, transposon-based mutagenesis, insertional mutagenesis, or both. In some embodiments, the set of desired properties comprises one or more of viability, growth rate, adaptability, doubling time, ratio of growth rate to genome size, ratio of doubling time to genome size, expression level of a gene of interest, and expression rate of a gene of interest.

In some embodiments, the first genome is viable. In some embodiments, N is an integer between 4 and 20. In some embodiments, the synthetic genome of interest is a minimal genome. In some embodiments, the second genome is smaller than the first genome in size. In some embodiments, the third genome comprises one or more fragments from a naturally occurring genome and one or more fragments from a synthetic genome. In step (b), the fragments of the first genome can be, in some embodiments, a nucleic acid molecule comprising one or more fragments of the first genome. For example, in step (d), the fragments of the first genome can be present in a single nucleic acid molecule before being combining with the fragment(s) of the second genome. In some embodiments, step (d) comprises deleting a portion or the entire fragment of the first genome that corresponds to one of the at least one fragment of the second genome. In some embodiments, one or more of the at least one fragment of the second genome is present in an extrachromosomal genetic element in the combining step (d). The extrachromosomal genetic element can be, for example, an episome, a plasmid, a fosmid, a cosmid, a bacterial artificial chromosome, or a yeast artificial chromosome. In some embodiments, the combining step comprises combining each of the two or more fragments of the second genome with fragments of the first genome to generate a plurality of the third genomes having all N corresponding fragments. In some embodiments, each of the plurality of the third genomes is tested for the set of desired properties.

In some embodiments, the combining step comprises chemically synthesizing and assembling the fragments of the first and second genomes to generate the third genome. In some embodiments, assembling the fragments of the first and second genomes comprises assembling chemically synthesized, overlapping oligonucleotides into one or more of nucleic acid cassettes. In some embodiments, a portion or the entire synthetic genome of interest is constructed from nucleic acid components that have been chemically synthesized, or that have been created from copies of the chemically synthesized nucleic acid components.

In some embodiments, the method further comprises modifying one or more genes in the third genomes after identifying the third genome as a synthetic genome of interest. In some embodiments, step (d) further comprises reorganizing gene order in the at least one fragment of the second genome before combining it with fragments of the first genome to generate the third genome. In some embodiments, the method further comprises reorganizing gene order in the third genome after it is identified as a synthetic genome of interest. In some embodiments, reorganizing gene order comprises grouping genes related to the same biological process in the at least one fragment of the second genome. In some embodiments, the same biological process is one or more of glucose transport and catabolism; ribosome biogenesis; protein export, DNA repair; transcription; translation; nucleotide synthesis, metabolism and salvage; glycolysis; metabolic processes; proteolysis; membrane transport; rRNA modification; and tRNA modification.

Also disclosed herein is a method for generating a viable genome of reduced size. In some embodiments, the method comprises: (a) providing a first genome known to be viable; (b) designing a second genome based on the first genome, wherein the second genome comprises a reduced number of genes of the first genome and is hypothesized to be viable; (c) dividing each of the first and second genomes into N corresponding fragments, wherein N is an integer equal to or greater than 3; (d) combining at least one of the N fragments of the second genome with a sufficient number of said fragments of the first genome to generate a third genome having all N corresponding fragments; (e) testing the third genome generated in step (d) for viability; (f) if the third genome is viable, identifying the third genome as a viable genome of reduced size; and (g) if the third genome is not viable, modifying one or more fragments of the second genome based on the testing of (e) and repeating steps (d)-(f) until the third genome is viable.

In some embodiments, the first genome is a naturally occurring genome. In some embodiments, the first genome is a genome of a unicellular organism. In some embodiments, the first genome is a bacterial genome, a yeast genome, a single-cell alga genome, or a combination thereof. In some embodiments, the first genome is a single chromosome genome. In some embodiments, the first genome is a multi-chromosome genome.

In some embodiments, the method further comprises deleting one or more genes from the third genomes after identifying the third genome as a viable genome of reduced size. In some embodiments, step (b) comprises testing the second genome for viability. In some embodiments, the method further comprises deleting one or more genes from at least one fragment of the second genome after identifying the third genome as a viable genome of reduced size and repeating steps (d)-(g) in one or more iterations.

In some embodiments, designing a second genome comprises modifying the first genome based on the information from literature resources, experimental data, or any combination thereof. In some embodiments, the experimental data comprises data obtained from a mutation study of the first genome, a genome related to the first genome, or any combination thereof.

In some embodiments, the experimental data comprises data related to genes of essential function redundancies (EFR). In some embodiments, the mutation study comprises one or more of transposon-based mutagenesis study, gene knockout study, and add-back study. In some embodiments, all of the N corresponding fragments are substantially the same length. In some embodiments, at least two of the N corresponding fragments are different in length. In some embodiments, at least one of the N corresponding fragments is a chromosome of the first or second genome. In some embodiments, at least one of the N corresponding fragments is a portion of a chromosome of the first or second genome.

In some embodiments, in step (d) the fragments of the first genome is a nucleic acid molecule comprising one or more fragments of the first genome. In some embodiments, step (d) comprises deleting a portion or the entire fragment of the first genome that corresponds to one of the at least one fragment of the second genome. In some embodiments, in the combining step (d), one or more of the at least one fragment of the second genome is present in an extrachromosomal genetic element. The extrachromosomal genetic element can be, for example, an episome, a plasmid, a fosmid, a cosmid, a bacterial artificial chromosome, or a yeast artificial chromosome.

In some embodiments, testing the genome for the set of desired properties comprises introducing the genome into a cell or a cell-like system. In some embodiments, the genome is introduced into the cell or the cell-like system by conjugation, transformation, transduction, or a combination thereof. In some embodiments, the cell-like systems comprises a membrane-bound volume, a lipid vesicle, a cell from which one or more intracellular components have been removed, a cell from which the resident genome has been removed, or any combination thereof.

In some embodiments, modifying at least one fragment of the second genome in step (f) is at least partly based on the testing of step (e). In some embodiments, modifying at least one fragment of the second genome fragments in step (f) further comprises conducting mutation study of the at least one fragment and modifying the at least one fragment at least partly based on the mutation study. The mutagenesis study can comprise, for example, random mutagenesis, targeted mutagenesis, or both. The mutageneis study comprises, in some embodiments, transposon-based mutagenesis, insertional mutagenesis, or both. In some embodiments, step (d) comprises chemically synthesizing and assembling the genomic fragments. In some embodiments, combining the fragments of the first and second genomes comprises assembling chemically synthesized, overlapping oligonucleotides into one or more of nucleic acid cassettes.

In some embodiments, the entire viable genome of reduced size is constructed from nucleic acid components that have been chemically synthesized, or that have been created from copies of the chemically synthesized nucleic acid components. In some embodiments, step (d) further comprises reorganizing gene order in the at least one fragment of the second genome before combining it with fragments of the first genome to generate the third genome. In some embodiments, the method further comprises reorganizing gene order in the third genome.

In some embodiments, reorganizing gene order comprises grouping genes related to the same biological process in the at least one fragment of the second genome. In some embodiments, the same biological process is one or more of glucose transport and catabolism; ribosome biogenesis; protein export, DNA repair; transcription; translation; nucleotide synthesis, metabolism and salvage; glycolysis; metabolic processes; proteolysis; membrane transport; rRNA modification; and tRNA modification.

In the methods and compositions disclosed herein, the first genome can have a size of no more than 15 Mb. In some embodiments, the first genome has a size of about 3 Mb to about 13 Mb.

Also disclosed are a synthetic genome produced by any of the methods disclosed herein, a synthetic cell produced by introducing the synthetic genome produced by any one of the methods disclosed herein into a cell-like system. In some embodiments, the cell-like system is a cell from which a resident genome has been removed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a non-limiting exemplary schematic illustration of a non-limiting embodiment of the design-build-test (DBT) cycle described herein for bacterial genomes.

FIGS. 2A-2C show the method used for creating eight NotI strains. FIG. 2A is a map of Syn1DREDIS genome showing the design of NotI restriction sites for the creation of 8 mycoides NotI strains. FIG. 2B is a schematic illustration showing that ⅛^(th) genome segments were released from mycoides genomes by restriction enzyme NotI and assembled in yeast. The three panels of FIG. 2C are gel photographs showing 8 NotI-digested genomic DNA (from 1 to 8) subjected to 1% agarose gel electrophoresis to separate ⅞^(th) and ⅛^(th) genome (top).

FIG. 3 is a non-limiting exemplary schematic illustration showing the structure of Tn5 puro transposon used for global mutagenesis, along with the sequence of Tn5 puro transposon.

FIG. 4 is a non-limiting exemplary schematic illustration showing exemplary steps in producing a Tn5 global insert library.

FIG. 5 is a non-limiting exemplary gene map showing that genes can be classified into 3 categories based on data from global Tn5 transposon mutagenesis.

FIG. 6 is a non-limiting exemplary Syn1.0 gene map showing the locations of Tn5 P4 insertions.

FIG. 7 shows the M. mycoides JCVI-Syn1.0 genome (1078 kb) displayed using CLC software.

FIG. 8 is a non-limiting exemplary diagram listing the genes deleted in the RGD2.0 design of segment 5.

FIG. 9 is a non-limiting exemplary Syn1.0 gene map showing the three design cycles involved in building Syn3.0.

FIG. 10 is a non-limiting exemplary schematic illustration showing the construction of the ⅛^(th) RGD+⅞ wild type genome by recombinase-mediated cassette exchange (RMCE).

FIGS. 11A-11G are non-limiting exemplary light micrographs showing that RGD1.0, Segment 6 (+⅞ Syn1.0) grew slowly and produced sectored colonies after 10 days. FIG. 11A is a light micrograph from transplantation of yeast clone #5 of RGD1.0 Segment 6+⅞ Syn1.0 at day 12. FIGS. 11B-11D are light micrographs from transplantation of yeast clone #6 of RGD1.0 Segment 6+⅞ Syn1.0 at day 10. FIGS. 11E-11G are light micrographs from transplantation of yeast clone #7 of RGD1.0 Segment 6+⅞ Syn1.0 at day 10.

FIG. 12 is a non-limiting exemplary sequence diagram showing that the mutations in Segment 6 sequence reduced the stability of a stem-loop transcription terminator downstream of the tRNA-His gene. At the top the relevant sequence region is shown for Syn1.0, and at the bottom is the segment 6 design sequence.

FIG. 13 is a non-limiting exemplary sequence diagram showing the mutations in Segment 6+⅞ in colony purified isolates from transplant of clone #5.

FIG. 14 is a non-limiting exemplary sequence diagram showing the mutations of tRNA-His terminator in RGD 1.0 Seg6+⅞ transplants (mutations at GC base pairs in the stem).

FIG. 15 is a non-limiting exemplary schematic illustration showing a non-limiting embodiment of the TREC-IN method.

FIGS. 16A-16B are non-limiting exemplary gel photographs analyzing twenty-four representative 1.4 kb dsDNA fragments assembled during the construction of HMG. FIG. 16A shows the dsDNA fragments following oligonucleotide assembly and amplification, and FIG. 16B shows the same dsDNA fragments following error correction and PCR amplification. Two microliters of each sample were loaded onto a 1% E-gel and run for 20 minutes. M indicates the 1 kb ladder (NEB).

FIG. 17 is a non-limiting exemplary diagram showing clone collapsing for scalable cassette identification. Twenty-four bacterial clones for each cassette were grown separately in liquid culture in 96-well plates and then collapsed into a single set of 24 wells. Each shaded grouping represents a single cassette while the shaded grouping in the last row is the final collapsed group.

FIGS. 18A-18B are non-limiting exemplary gel photographs showing rolling circle amplification (RCA) products derived from the HMG eighth molecule assemblies. FIG. 18A shows supercoil DNA extracted from yeast clones containing the HMG eighth molecule assemblies and used as template in RCA reaction with GE-Templiphi Large Construct kit. FIG. 18B shows supercoil DNA extracted from yeast clones containing the HMG eighth molecule assemblies and used as template in RCA reaction with Qiagen-REPLI-g kit.

FIG. 19 is a non-limiting exemplary gel photograph showing field-inverted gel electrophoresis analysis of HMG.

FIG. 20 is a non-limiting exemplary schematic illustration showing the editing of previously generated sequence-verified cassettes.

FIG. 21 is a non-limiting exemplary schematic illustration showing the strategy for whole genome synthesis.

FIGS. 22A-22B show the three gene classifications based on Tn5 mutagenesis data. FIG. 22A is a gene map showing examples of the 3 gene classifications based on Tn5 mutagenesis data. FIG. 22B is a pie chart showing the number of Syn1.0 genes in each Tn5 mutagenesis classification group. n-genes and in-genes were candidates for deletion in reduced genome designs.

FIG. 23 is a non-limiting exemplary schematic illustration showing the TREC deletion method.

FIG. 24 is a non-limiting exemplary schematic illustration showing genome engineering to produce the Syn2.0 in yeast.

FIG. 25 is a non-limiting exemplary Syn1.0 gene map showing the three DBT cycles involved in building Syn3.0.

FIG. 26 is a non-limiting exemplary BLAST map showing proteins in Syn3.0 and homologs found in other organisms.

FIG. 27 is a non-limiting exemplary pie chart showing the partition of genes into four major functional groups.

FIGS. 28A-28D compare Syn1.0 and Syn3.0 growth features. The two panels of FIG. 28A are light micrographs comparing colony sizes and morphologies of Syn1.0 and Syn3.0 cells derived from 0.2 μm-filtered liquid cultures diluted and plated on agar medium for 96 h (scale bars=1.0 mm). FIG. 28B is a plot of fluorescent measure (RFU) vs. time showing the growth rates in liquid static culture determined using a RFU of dsDNA accumulation over time to calculate doubling times (td). The panels of FIG. 28C are differential interference contrast micrographs showing native cell morphology in liquid culture imaged in wet mount preparations (scale bars=10 μm). Panels of FIG. 28D are scanning electron micrographs of Syn1.0 (left, scale bars=200 nm) and Syn3.0 (middle, scale bars=200 nm and right, scale bars=1 μm). The panel on the right shows a variety of the structures observed in Syn3.0 cultures.

FIG. 29 is a non-limiting exemplary plot of RFU vs. cell concentration showing the correlation of PicoGreen fluorescence with cell concentration.

FIG. 30 is a non-limiting illustrative diagram showing the reorganization of gene order in segment 2.

FIGS. 31A-31B show the testing of gene content and codon usage principles using the DBT cycle. FIG. 31A is a diagram of the modified rrs gene showing its secondary structure that was successfully incorporated into the Syn3.0 genome carrying M. capricolum mutations and h39 (inset) swapped with that of E. coli. FIG. 31B shows that three different codon optimization strategies were used for modifying the sequence of the essential genes era, recO and glyS by using M. mycoides codon adaption index (CAI) or that of E. coli with the codons TGG or TGA encoding tryptophan.

FIG. 32 is a non-limiting exemplary plot showing recovery of cells following electroporation. Cells were allowed to recover in YPDS medium at 30° C. and plated at intervals on CAA-URA medium. FIG. 32 shows that cell viability increased nearly 10-fold in the interval from 8 to 10 hours, and thereafter the cell number increased at about the doubling rate.

FIG. 33 is a non-limiting exemplary plot of a Section of the K. marxianus Chromosome 7 ScURA3 insertion map

FIG. 34A-34B is a non-limiting exemplary schematic illustration of a proposed NHEJ insertion mechanism to explain the observed types of ScURA3/genome junctions.

FIG. 35 is a non-limiting exemplary map of pCC1BAC-LCyeast_(scHis3)-SYN-KM_CENARS_oriT. oriT sequence was inserted between EcoRI and BamHI.

FIG. 36 shows establishment of E. coli to K. marxianus conjugation method. E. coli (EPI300) was transformed with plasmids in the bottom table.

FIG. 37 shows non-limiting exemplary gel electrophoresis photographs of TAE+EtBr gelsconfirming the presence of pCC1BAC-LCyeast_(scHis3)-SYN-KM_CENARS_oriT in K. marxianus. Eight K. marxianus conjugation colonies were screened for the presence of pCC1BAC-LCyeast_(scHis3)-SYN-KM_CENARS_oriT. Genomic DNA (left panel); oriT PCR product (right panel).

FIG. 38 is a non-limiting exemplary gel electrophoresis photograph showing that large DNA fragment can be transferred from E. coli to K. marxianus via conjugation. Lane 1. #4-55—clone 1; Lane 2. #4-55—clone 2; Lane 3. #4-55—E. coli; Lane 4. #3; Lane 5. NEB 1 kb ladder. (genomic DNA on TAE gel with SYBR-gold Staining).

FIG. 39 is a non-limiting exemplary schematic illustration of a design-build-test cycle in K. marxianus.

FIG. 40 shows a non-limiting exemplary gel electrophoresis photograph illustrating transfer and stable maintenance of Stage-II molecule into #6_12 in K. marxianus. Stage-II molecule #6_12 was visualized after transformation and growth in selective media for several generations. Controls—#6_12 extracted from E. coli and S. cerevisiae.

FIG. 41 shows non-limiting exemplary gel electrophoresis photographs illustrating colony PCR analysis of CRISPR/Cas9 mediated chromosomal deletion. 96 transformants and a wild-type (wt) control were screened using primers. WT PCR was loaded on the last lane of each row (24 samples each). Control colony produced 300 bp and 400 bp PCR amplicons, while none of the 96 transformants did so. Instead, the transformants produced a ˜465 bp PCR amplicon, indicative of a ˜80 kb deletion of the chromosomal DNA corresponding to the #6_12 episomal DNA.

FIG. 42 is a non-limiting exemplary gel electrophoresis photograph verifying the presence of the #6_12 episomal DNA in six K. marxianus strains that lack the corresponding segment from the chromosome. A mixture of 80 kb and 90 kb supercoiled DNA was loaded as a size-estimating control followed by DNA extracted from the transformants 1-6, deemed positive by colony PCR (FIG. 41).

FIG. 43 shows a non-limiting exemplary qPCR design.

FIG. 44 is a non-limiting exemplary plot showing results of using qPCR to determine the copy number of #6_12 DNA fragment in the wild-type strain (WT), strain carrying episomal DNA #6_12 (Episome+chr) and strain carrying episomal DNA #6_12 but with the corresponding chromosomal fragment deleted (Episome only).

FIG. 45 is a non-limiting exemplary pulsed-field gel electrophoresis (PFGE) photograph confirming ˜80 kb deletion from the chromosome in the strain carrying the episome #6_12. Lane 1—wild-type strain (WT), lane 2—strain carrying episomal DNA #6_12 (Episome+chr), lane 3—strain carrying episomal DNA #6_12 but with the corresponding chromosomal fragment deleted (Episome only). Chromosome 7 with the 80 kb deletion migrates faster (lane 3) than the corresponding wild-type chromosome (lanes 1 and 2).

FIG. 46 is a non-limiting exemplary gel electrophoresis photograph showing that minimized sub-chromosomal molecule #2_37 was introduced into K. marxianus using conjugation. Stable maintenance of this episome was verified by growing the transformed cells in selective media, extracting DNA and resolving the DNA alongside the same episomal molecule extracted from E. coli.

FIG. 47 show non-limiting exemplary gel electrophoresis photographs showing colony PCR analysis of CRISPR/Cas9 mediated chromosomal deletion. 48 transformants and a wild-type (wt) control were screened using primers. Wt PCR was loaded on the last lane of each row (24 samples each). Control colony produced 350 bp and 450 bp PCR amplicons, while none of the 48 transformants did so. Instead, several transformants produced a single ˜360 bp PCR amplicon, indicative of a ˜91 kb deletion of the chromosomal DNA corresponding to the #2_37 minimized episomal DNA.

FIG. 48 is a non-limiting exemplary schematic illustration showing that primers for three amplicons were designed such that two amplicons would be from different parts of the segment encompassed by #2_37 (amplicons 1-2) and one outside this segment (amplicon 3). qPCR would help determine the relative copy number of the DNA fragment encoded in #2_37 fragment. In the wild-type strain, amplicons 1-2 should be of the same relative amount in a qPCR as amplicon 3; when the episomal DNA #2_37 was introduced, the cell should carry two copies of parts of the segment encoded in #2_37—one in the chromosome, another in the episome: this would result in twice amount of amplicons 1-2 relative to 3; however after the CRISPR/Cas9 mediated deletion, the copy number of segment #2_37 returned to one since there is only one copy of this DNA fragment—in the episome: this would result in the same relative amount of amplicons 1-2 compared to amplicon 3. Amplicons 1-2: probe; Amplicon 3: Control.

FIG. 49 is a non-limiting exemplary plot showing that qPCR was used to determine the copy number of #2_37 DNA fragment in the wild-type strain (WT), strain carrying the minimized episomal DNA #2_37 (Episome+chr) and strain carrying the minimized episomal DNA #2_37 but with the corresponding wildtype chromosomal fragment deleted (Episome only).

FIG. 50 is a non-limiting exemplary pulsed-field gel electrophoresis (PFGE) photograph confirming that ˜91 kb deletion from the chromosome in the strain carrying the minimized episome #2_37. Lane 1—wild-type strain (WT), lane 2—strain carrying minimized episomal DNA #2_37 (Episome+chr), lane 3—strain carrying minimized episomal DNA #2_37 but with the corresponding wild-type chromosomal fragment deleted (Episome only). Chromosome 7 with the ˜91 kb deletion migrates faster (lane 3) than the corresponding wild-type chromosome (lanes 1 and 2).

DETAILED DESCRIPTION

In the following detailed description, reference is made to the accompanying drawings, which form a part hereof. In the drawings, similar symbols typically identify similar components, unless context dictates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure, as generally described herein, and illustrated in the Figures, can be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are explicitly contemplated herein.

All patents, published patent applications, other publications, and sequences from GenBank, and other databases referred to herein are incorporated by reference in their entirety with respect to the related technology.

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. See, e.g. Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). For purposes of the present disclosure, the following terms are defined below.

As used herein, the terms “nucleic acid,” “nucleic acid molecule”, and “oligonucleotide” and “polynucleotide” are used interchangeably. Examples of nucleic acids include, but are not limited to, deoxyribonucleic acid (DNA); ribonucleic acid (RNA); modified nucleic acid molecules such as peptide nucleic acid (PNA), locked nucleic acids (LNA); cDNA; genomic DNA, mRNA, synthetic nucleic acid molecule (such as that are chemically synthesized or recombinantly produced), and any combination thereof. Nucleic acid molecules can be double-stranded or single-stranded. Where single-stranded, the nucleic acid molecule can be the sense strand or the antisense strand. The nucleic acid molecules can be circular or linear.

As used herein, the term “genome” refers to whole (complete) genome and portions of whole genomes having nucleic acid sequences sufficient to effect and/or sustain viability of a cell (minimal cellular genome), of an organism that depends on a host cell for viability (e.g., minimal viral genome), or organelle function within a host cell (minimal organelle genome) under at least one set of culturing or environmental conditions. The genome can be a viral genome, a genome of organelles (e.g., mitochondria or chloroplast), and a genome of self-replicating organisms (e.g., cellular organisms, including, but not limited to, prokaroytes and eukaryotes). For example, the genome can be a genome of bacteria (e.g., Mycoplasma), yeast (e.g., S. cerevisiae and K. marxianus), archebacteria, vertebrates, or mammals. A genome can also be an entirely new construct for an organism that does not fall into any known Linnean category. In some embodiments, the genome may be a genome of a microorganism, such as a unicellular microorganism (e.g., a bacterium and yeast). In some embodiments, the genes in a genome may be in the order found in the microorganism, or they may be shuffled. A genome may also include mutant versions of one or more of the genes contained therein.

As used herein, a “cellular genome” or a “synthetic cellular genome” refers to a genome that comprises sequences which encode and may express nucleic acids and proteins required for some or all of the processes of transcription, translation, energy production, transport, production of cell membranes and components of the cell cytoplasm, DNA replication, cell division, and the like. A “cellular genome” differs from a viral genome or the genome of an organelle, at least in that a cellular genome contains the information for replication of a cell, whereas viral and organelle genomes contain the information to replicate themselves (sometimes with the contribution of cellular factors), but they lack the information to replicate the cell in which they reside.

As used herein, a “foreign gene” or a “foreign genome” is a gene or genome derived from a source other than the resident (original) organism, e.g., from a different species of the organism.

As used herein, the term “membrane-bound vesicle” refers to a vesicle in which a lipid-based protective material encapsulates an aqueous solution.

As used herein, the term “minimal genome,” with respect to a cell, refers to a genome consisting of or consisting essentially of a minimal set of genetic sequences that are sufficient to allow for cell survival under specified environmental (e.g., nutritional) conditions. A “minimal genome,” with respect to an organelle, as used herein, refers to a genome consisting of or consisting essentially of a minimal set of genetic sequences that are sufficient to allow the organelle to function. A minimal genome must contain sufficient information to allow the cell or organelle to carry out essential biological processes, such as, for example, transcription, translation, use of an energy source, transport of salts, nutrients and the like into and out of the organelle or cell, etc. A “minimal replicating genome,” with respect to either a cell or an organelle, contains, in addition, genetic sequences sufficient to allow for self-replication of the cell or organelle. Thus, a “minimal replicating synthetic genome” is a single polynucleotide or group of polynucleotides that is at least partially synthetic and that contains the minimal set of genetic sequences for a cell or organelle to survive and replicate under specific environmental conditions.

As used herein, a “synthetic genome” includes a single polynucleotide or group of polynucleotides that contain the information for a functioning organelle or organism to survive and, optionally, replicate itself where particular environmental (e.g., nutritional or physical) conditions are met. All or at least part of the genome (e.g., a cassette) is constructed from components that have been chemically synthesized, or from copies of chemically synthesized nucleic acid components. The copies may be produced by any of a variety of methods, including cloning and amplification by in vivo or in vitro methods. In one embodiment, an entire genome is constructed from nucleic acid that has been chemically synthesized, or from copies of chemically synthesized nucleic acid components. Such a genome is sometimes referred to herein as a “completely synthetic” genome. In other embodiments, one or more portions of the genome may be assembled from naturally occurring nucleic acid, nucleic acid that has been cloned, or the like. Such a genome is sometimes referred to herein as a “partially synthetic” or “semi-synthetic” genome.

As used herein, the term “cell-like system” refers to a system that resembles a naturally occurring cell, but does not occur without human intervention. Non-limiting examples of cell-like systems include mammalian red blood cells (mammalian red blood cells do not naturally contain a genome) into which a genome or partial genome has been installed (or “introduced”); a “ghost” cell into which a foreign genome has been introduced; an aqueous volume enclosed by a phospholipid bilayer (whether derived from a naturally occurring cell membrane, manmade, or a hybrid of naturally occurring and manmade components) into which a genome has been introduced; and an aqueous volume enclosed by a lipid vesicle into which a genome has been introduced. As used herein, a “ghost cell” is a cell that naturally encloses a genome, but from which the naturally occurring genome is absent either as a result of genetic programming causing some cells to be genome-free or because the genome has been removed or inactivated. A naturally occurring genome may be removed from a cell by various methods, for example, by lysis and digestion, as described in US20070269862 (the content of which is incorporated hereby in its entirety). Ghost cells can also be produced by means, including but not limited to physical methods such ultraviolet and gamma irradiation, genetic methods involving minicells, and treatment with chemical compounds such as antibiotics and peroxides. In a non-limiting exemplary embodiment, the naturally occurring genomes are removed from a cell of Mycoplasma mycoides (M. mycoides), and a synthetic M. mycoides genome of reduced size may be introduced into the M. pneumoniae ghost cell. In some embodiments, ghost cells are produced from yeast (e.g., Kluyveromyces marxianus (K. marxianus)), and a synthetic K. marxianus genome of reduced size may be introduced into the K. marxianus ghost cell.

The ability to design and produce a synthetic genome, and generate a cell or cell-like system including the synthetic genome along with a membrane and cytoplasm or membrane-bound aqueous volume, is very valuable in the fields like cell biology and biotechnology. In the present disclosure, methods for generating synthetic genomes, for example synthetic genomes having desired properties and viable genomes of reduced size, are disclosed. In some embodiments, the methods include designing a synthetic genome of interest; building the genome of interest through, for example, dividing and combining fragments of various parent genomes; and testing the resulting genome of interest. Such a design-build-test procedure can be iterated for one or more times, for example until a desired synthetic genome of interest is obtained. A non-limiting schematic illustration of the design-build-test (DBT) cycle described herein for bacterial genomes is provided in FIG. 1. The main design objective for the DBT cycle shown in FIG. 1 is genome minimization. As an example, in a DBT cycle, starting from a first genome (e.g., a naturally occurring genome), a reduced genome (i.e., a second genome) is designed by removing non-essential genes (e.g., genes determined as non-essential by global transposon (e.g., Tn5) gene disruption) from the first genome. Each of the first and second genomes is divided into 8 corresponding genomic fragments, and one or more of the genomic fragments of the second genome is combined with the genomic fragments of the first genome to generate a third genome having all 8 corresponding genomic fragments. The third genome is tested for one or more properties (e.g., phenotypes), including but not limited to, viability, growth rate, adaptability, doubling time, ratio of growth rate to genome size, ratio of doubling time to genome size, expression level of a gene of interest, and expression rate of a gene of interest. For example, each of the 8 corresponding genomic fragments from the second genome can be combined with the genomic fragments of the first genome to generate a third genome having all 8 corresponding genomic fragments, and thus all together 8 different third genomes can be produced and each of the third genomes can be tested for the properties. Before the genomic fragment(s) of the second genome combine with the genomic fragment(s) of the first genome, one or more of the genomic fragments (from the second and/or the first genome) can be modified, for example, by deleting or adding one or more genes or non-coding regions. In some embodiments, after the genomic fragment(s) of the second genome combine with the genomic fragment(s) of the first genome, one or more of the genomic fragments (from the second and/or the first genome) can be modified, for example, by deleting or adding one or more genes or non-coding regions. In some embodiments, the modification can be preformed both before and after combining the genomic fragments of the first and the second genomes. At each DBT cycle, gene essentiality can be re-evaluated, for example, by transposon (e.g., Tn5) mutagenesis.

Also disclosed herein are synthetic genomes produced by the methods disclosed herein, synthetic cells containing the synthetic genomes, and the methods for producing the synthetic cells.

In some embodiments, the method for generating a synthetic genome of interest comprises: (a) providing a first genome; (b) designing a second genome based on the first genome, wherein the second genome is hypothesized to have a set of desired properties; (c) dividing each of the first and second genomes into N corresponding fragments, wherein N is a positive integer; (d) combining at least one fragment of the second genome with fragments of the first genome to generate a third genome having all N corresponding fragments; and (e) testing the third genome generated in step (d) for the set of desired properties. In some embodiments, the method can also comprise (f) identifying the third genome as a synthetic genome of interest if it has the set of desired properties; otherwise modifying at least one fragment of the second genome and repeating steps (d)-(f) in one or more iterations until a set of desired properties is obtained in the third genome. The method can be used to produce genome with various desired properties. Non-limiting examples of the desired properties include one or more of viability, growth rate, adaptability, doubling time, ratio of growth rate to genome size, ratio of doubling time to genome size, expression level of a gene of interest, and expression rate of a gene of interest, ratio of viability to genome size, ratio of viability to expression level of a gene of interest, ratio of growth rate of expression level of a gene of interest, and ratio of growth rate to expression level of a gene of interest. In some embodiments, the first genome is a viable genome. As used herein, a viable cellular genome refers to a cellular genome that contains nucleic acid sequences sufficient to cause and/or sustain viability of a cell, e.g., those encoding molecules required for replication, transcription, translation, energy production, transport, production of membranes and cytoplasmic components, and cell division. In some embodiments, the first genome is a naturally occurring genome.

The second genome can be smaller or larger than the first genome in size. In some embodiments, the second genome has the same size as the first genome. In some embodiments, the synthetic genome of interest is a genome of reduced size, for example a minimal genome. The doubling time for the minimal genome can vary, for example from about 1 hour to about 10 days. In some embodiments, the doubling time for the minimal genome can be, or be about, 1 hour, 5 hours, 10 hours, 15 hours, 20 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or a range between any two of these values. In some embodiments, the doubling time for the minimal genome can be about 4 days.

The methods disclosed herein can be used to generate a viable genome of reduced size. In some embodiments, the method comprises: (a) providing a first genome known to be viable; (b) designing a second genome based on the first genome, wherein the second genome comprises a reduced number of genes of the first genome and is hypothesized to be viable; (c) dividing each of the first and second genomes into N corresponding fragments, wherein N is a positive integer; (d) combining at least one said fragment of the second genome with a sufficient number of said fragments of the first genome to generate a third genome having all N corresponding fragments; (e) testing the third genome generated in step (d) for viability; (f) if the third genome is viable, identifying the third genome as a viable genome of reduced size; and (g) if the third genome is not viable, modifying one or more fragments of the second genome based on the testing of (e) and repeating steps (d)-(f) until the third genome is viable. In some embodiments, the method further comprises deleting one or more genes or non-coding regions from at least one fragment of the second genome after identifying the third genome as a viable genome of reduced size and repeating step (d)-(g) in one or more iterations. In some embodiments, the method further comprises deleting one or more genes or non-coding regions from the third genome after identifying the third genome as a viable genome of reduced size.

In the methods disclosed herein, in combining step (d) the fragments of the first genome can be present in a single nucleic acid molecule. In some embodiments, step (d) comprises combining a fragment of the second genome with the entire or substantial portion of the first genome. Step (d) can, for example, comprise deleting a portion or the entire fragment of the first genome that corresponds to the fragment of the second genome that is combined with the entire or substantial portion of the first genome. In some embodiments, the deletion comprises replacing the portion or the entire fragment of the first genome with the corresponding fragment of the second genome. In some embodiments, it can be advantageous to delete only a portion (for example a half) of the fragment of the first genome to allow identification of the portion of the fragment responsible for the tested properties or the lack of tested properties. The tested properties can comprise, for example, viability. The deletion of genomic fragment(s) can be achieved using any suitable methods known in the art, for example, recombinase-mediated homologous recombination, CRISPR/Cas9 mediated deletion, or a combination thereof. The recombinase can be, for example, Cre-recombinase. As disclosed herein, the fragment(s) of the second genome can be present in an extrachromosomal genetic element before they are combined with the fragment(s) of the first genome to generate the third genome, after they are combined with the fragment(s) of the first genome to generate the third genome, or both. The third genome can comprise one or more extrachromosomal genetic elements. Non-limiting examples of the extrachromosomal genetic element include episomes, plasmids, fosmids, cosmids, bacterial artificial chromosomes, yeast artificial chromosomes, or any combination thereof. In some embodiments, the third genome comprises at least one chromosome comprising both the fragment(s) of the second genome and the fragment(s) of the first genome.

In the methods disclosed herein, the value of N can vary. For example, N can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range between any two of these values (including end points). In some embodiments, N is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or larger. In some embodiments, N can be an integer equal to or greater than 3. In some embodiments, N is an integer between 4 and 20. In some embodiments, N is 8. In addition, the value of N can vary in the iterations when steps (d)-(f) are performed. The value of N can be different for each of the iterations when steps (d)-(f) are performed, or the value of N can be different for some of the iterations when steps (d)-(f) are performed. For example, the value of N may be 8 in the first iteration and the value of N may be smaller (e.g., 3, 4, 5, 6, or 7) or larger than 8 (e.g., 9, 10, 11, or 12) in the second iteration when steps (d)-(f) are repeated. As another non-limiting example, N may be 8 in the first iteration, 10 in the second iteration and 12 in the third iteration. In yet another non-limiting example, N may be 9 in the first iteration, 12 in the second iteration, and 9 in the third iteration. As another non-limiting example, for a multi-chromosome genome (e.g., the first genome, the second genome, the synthetic genome of interest), one chromosome of the genome can be divided into a number of fragments and the other chromosomes of the genome can be considered as one fragment or multiple fragments. For example, for a K. marxianus genome having 8 chromsomes, Chromosome No. 7 (“chromosome 7”) can be divided into 12 fragments, and the remaining seven chromsomes can be considered as one fragment. Therefore, each of a first and second K. marxianus genome can be divided into 13 corresponding fragments. One or more of the 1/12^(th) of Chromosome No. 7 of the second K. max genome can, for example, be combined with fragments of the second K. marxianus genome to generate a third K. marxianus genome having all 13 corresponding fragments.

In some embodiments, one chromosome of a multi-chromosome genome having Z chromosomes (Z is a positive integer >=2) is divivided into M fragments (M is a positive integer >=3), and the one chromosome is divided into (M-Z+1) fragments. One or more (e.g., each of) of the (M-Z+1) fragments of the one chromosome (referred to as “the test subchromosomal molecule”) can be modified (e.g., by deletion, addition, substitution, or a combination thereof, of one or more genes or non-coding regions) tested entirely or in a portion (e.g., one half) at a time. For example, introduce the test subchromosomal molecule can be encoded in an episome and combine with fragments of the multi-chromosome genome to generate a third genome for testing. In some embodiments, the corresponding chromosomal segment of the multi-chromosome genome can be deleted entirely, for example using CRISPR/Cas9, to test the functionality of the introduced test subchromosomal molecule. In some embodiments, only a portion of the corresponding chromosomal segments (for example, one half) is deleted for testing, which can allow identification of specific region in the test subchromosomal molecule that result in an observed property (e.g., viability). For example, if a minimized subchromosomal molecule is non-functional, this method can be helpful in determining which part of the minimized molecule resulted in a non-viable phenotype. In some embodiments, direct swapping of the chromosomal segment can be used. For example, using a selectable auxotrophic marker, the test subchromosomal molecule can be directly swapped for the corresponding wild-type chromosomal fragment and tested for one or more desired properties (e.g., viability). In some embodiments, the swapping of chromosomal fragments can be achieved using recombinase-mediated homolgous recombination event. For example, loxP sites can be added to the test subchromosomal molecule and at the corresponding locations in the wildtype chromosome to enable Cre-recombinase mediated “swapping” event.

The N corresponding fragments can be of the same or different length. For example, all of the N corresponding fragments can be the same length, or be substantially the same length. As used herein, two genomic fragments are considered to be substantially the same length if the difference in their length is no more than 10% of the entire length of the genomic fragment that is longer. The N corresponding fragments can also be different in length. For example, two or more of the N corresponding fragments may be different in length. In some embodiments, each of the N corresponding fragments is different in length. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight of the N corresponding fragments are different in length.

Each of the N corresponding fragments can be a portion of the genome (e.g., a portion of the first, the second, or the third genome). A portion of a genome can be, for example, a portion of a chromosome of the genome, a chromosome of the genome, two or more chromosomes of the genome, or a portion of a chromosome of the genome as well as one or more remaining chromosomes of the genome. In some embodiments, a portion of the genome can be one or more chromosomes, one or more chromosome fragments, or any combination thereof. For example, a portion of the genome may be any fraction of a naturally occurring genome, one or more fragments of one or more naturally occurring chromosomes, one or more fragments of one or more naturally occurring chromosomes and one or more manmade nucleic acid sequences, one or more manmade nucleic acid sequences or fragments of manmade nucleic acid sequences, or any combination thereof. For example, for a single-chromosome genome, one of the N corresponding fragments can be, or be about, 1/N of the genome; or longer than or shorter than 1/N of the genome. As another example, for an eight-chromosome genome, one of the N corresponding fragments can be two of the eight chromosomes of the genome or 1/12th of one of the eight chromosomes (e.g., Chromosome No. 7), or the fragment can be one and a half of the eight chromosomes of the genome.

One or more of the N corresponding fragments of a genome may overlap with one or more of the remaining genomic fragments of the genome. For example, if the first genome is divided into four fragments, the first fragment may overlap with the second and the fourth fragment at the 5′ and 3′ terminus, respectfully; the second fragment may overlap with the third and the first fragment at the 5′ and 3′ terminus, respectfully; the third fragment may overlap with the second and the four fragment at the 5′ and 3′ terminus, respectfully; and the fourth fragment may overlap with the third and the first fragment at the 5′ and 3′ terminus, respectfully. It may also be that one or more of the fragments only overlap with one fragment (e.g., the second fragment), but not overlap with other fragments. The overlapping between two genomic fragments can vary in length. For example, the length of the overlapping can be 1 bp to 100 kb, or longer. In some embodiments, the overlapping between two genomic fragments is, or is about, 1 bp, 10 bp, 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 5 kb, 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, or a range between any two of these values.

One or more of the N corresponding genomic fragments of the first genome and/or the second genome can be modified before being combined to generate the third genome. In some embodiments, only fragment(s) of the first genome is modified before being combined to generate the third genome, and none of the fragments of the second genome is not modified. For example, one, two, three, four, or more of the fragments of the first genome are modified before being combined with the fragments of the second genome to generate the third genome. In some embodiments, only fragment(s) of the second genome is modified before being combined to generate the third genome, and none of the fragments of the first genome is not modified. For example, one, two, three, four, or more of the fragments of the second genome are modified before being combined with the fragments of the first genome to generate the third genome. In some embodiments, at least one fragment of the first genome and at least one fragment of the second genome are modified before being combined with other fragments of the first and second genome to generate the third genome. The genomic fragments can be modified based on information from various sources as described herein, for example, including but not limited to, knowledge known in the art, scientific publications, experimental data, and any combination thereof.

The type of the genome (e.g., the first genome, the second genome, the third genome, and the genome of interest) can vary. For example, the genome can be a viral genome, an organelle genome, a genome from a unicellular (i.e., single-cell) organism, or a genome from a multicellular organism. In some embodiments, the genome is a prokaryotic genome. In some embodiments, the genome is a eukaryotic genome. Examples of the genome includes, but are not limited to, Aeropyrum pernix; Agrobacterium tumefaciens; Anabaena; Anopheles gambiae; Apis mellifera; Aquifex aeolicus; Arabidopsis thaliana; Archaeoglobus fulgidus; Ashbya gossypii; Bacillus anthracis; Bacillus cereus; Bacillus halodurans; Bacillus licheniformis; Bacillus subtilis; Bacteroides fragilis; Bacteroides thetaiotaomicron; Bartonella henselae; Bartonella quintana; Bdellovibrio bacteriovirus; Bifidobacterium longum; Blochmannia floridanus; Bordetella bronchiseptica; Bordetella parapertussis; Bordetella pertussis; Borrelia burgdorferi; Bradyrhizobium japonicum; Brucella melitensis; Brucella suis; Buchnera aphidicola; Burkholderia mallei; Burkholderia pseudomallei; Caenorhabditis briggsae; Caenorhabditis elegans; Campylobacter jejuni; Candida glabrata; Canis familiaris; Caulobacter crescentus; Chlamydia muridarum; Chlamydia trachomatis; Chlamydophila caviae; Chlamydophila pneumoniae; Chlorobium tepidum; Chromobacterium violaceum; Ciona intestinalis; Clostridium acetobutylicum; Clostridium perfringens; Clostridium tetania Corynebacterium diphtheriae; Corynebacterium efficiens; Coxiella burnetii; Cryptosporidium hominis; Cryptosporidium parvum; Cyanidioschyzon merolae; Debaryomyces hansenii; Deinococcus radiodurans; Desulfotalea psychrophila; Desulfovibrio vulgaris; Drosophila melanogaster; Encephalitozoon cuniculi; Enterococcus faecalis; Erwinia carotovora; E. coli; Fusobacterium nucleatum; Gallus gallus; Geobacter sulfurreducens; Gloeobacter violaceus; Guillardia theta; Haemophilus ducreyi; Haemophilus influenzae; Halobacterium; Helicobacter hepaticus; Helicobacter pylori; Homo sapiens; Kluyveromyces sp; Kluyveromyces marxianus; Kluyveromyces waltii; Lactobacillus johnsonii; Lactobacillus plantarum; Legionella pneumophila; Leifsonia xyli; Lactococcus lactis; Leptospira interrogans; Listeria innocua; Listeria monocytogenes; Magnaporthe grisea; Mannheimia succiniciproducens; Mycoplasma forum; Mesorhizobium loti; Methanobacterium the rmoautotrophicum; Methanococcoides burtonii; Methanococcus jannaschii; Methanococcus maripaludis; Methanogenium frigidum; Methanopyrus kandleri; Methanosarcina acetivorans; Methanosarcina mazei; Methylococcus capsulatus; Mus musculus; Mycobacterium Bovis; Mycobacterium leprae; Mycobacterium paratuberculosis; Mycobacterium tuberculosis; Mycoplasma gallisepticum; Mycoplasma genitalium; Mycoplasma mycoides; Mycoplasma penetrans; Mycoplasma pneumoniae; Mycoplasma pulmonis; Mycoplasma mobile; Nanoarchaeum equitans; Neisseria meningitidis; Neurospora crassa; Nitrosomonas europaea; Nocardia farcinica; Oceanobacillus iheyensis; Onions yellows phytoplasma; Oryza sativa; Pan troglodytes; Pasteurella multocida; Phanerochaete chrysosporium; Photorhabdus luminescens; Picrophilus torridus; Plasmodium falciparum; Plasmodium yoelii yoelii; Populus trichocarpa; Porphyromonas gingivalis Prochlorococcus marinus; Propionibacterium acnes; Protochlamydia amoebophila; Pseudomonas aeruginosa; Pseudomonas putida; Pseudomonas syringae; Pyrobaculum aerophilum; Pyrococcus abyssi; Pyrococcus furiosus; Pyrococcus horikoshii; Pyrolobus fumarii; Ralstonia solanacearum; Rattus norvegicus; Rhodopirellula baltica; Rhodopseudomonas palustris; Rickettsia conorii; Rickettsia typhi; Rickettsia prowazekii; Rickettsia sibirica; Saccharomyces cerevisiae; Saccharomyces bayanus; Saccharomyces boulardii; Saccharopolyspora erythraea; Salmonella enterica; Salmonella typhimurium; Schizosaccharomyces pombe; S. cerevisiae; Shewanella oneidensis; Shigella flexneria; Sinorhizobium meliloti; Staphylococcus aureus; Staphylococcus epidermidis; Streptococcus agalactiae; Streptococcus mutans; Streptococcus pneumoniae; Streptococcus pyogenes; Streptococcus thermophilus; Streptomyces avermitilis; Streptomyces coelicolor; Sulfolobus solfataricus; Sulfolobus tokodaii; Synechococcus; Synechocystis; Takifugu rubripes; Tetraodon nigroviridis; Thalassiosira pseudonana; Thermoanaerobacter tengcongensis; Thermoplasma acidophilum; Thermoplasma volcanium; The rmosynechococcus elongatus; Thermotagoa maritima; Thermus thermophilus; Treponema denticola; Treponema pallidum; Tropheryma whipplei; Ureaplasma urealyticum; Vibrio cholerae; Vibrio natriegens; Vibrio parahaemolyticus; Vibrio vulnificus; Vibrio species: adaptatus, aerogenes, aestivus, aestuarianus, agarivorans, albensis, alfacsensis, alginolyticus, anguillarum, areninigrae, artabrorum, atlanticus, atypicus, azureus, brasiliensis, bubulus, calviensis, campbellii, casei, chagasii, cholera, cincinnatiensis, coralliilyticus, crassostreae, cyclitrophicus, diabolicus, diazotrophicus, ezurae, fischeri, fluvialis, fortis, furnissii, gallicus, gazogenes, gigantis, halioticoli, harveyi, hepatarius, hippocampi, hispanicus, hollisae, ichthyoenteri, indicus, kanaloae, lentus, litoralis, logei, mediterranei, metschnikovii, mimicus, mytili, natriegens, navarrensis, neonates, neptunius, nereis, nigripulchritudo, ordalii, orientalis, pacinii, parahaemolyticus, pectenicida, penaeicida, pomeroyi, ponticus, proteolyticus, rotiferianus, ruber, rumoiensis, salmonicida, scophthalmi, splendidus, superstes, tapetis, tasmaniensis, tubiashii, vulnificus, wodanis, and xuii; Wigglesworthia glossinidia; Wolbachia pipientis; Wolinella succinogenes; Xanthomonas axonopodis; Xanthomonas campestris; Xylella fastidiosa; Yarrowia lipolytica; Yersinia pseudotuberculosis; and Yersinia pestis.

Other examples of genomes include, but are not limited to any microorganism of the class Labyrinthulomycetes. While the classification of the Thraustochytrids and Labyrinthulids has evolved over the years, for the purposes of the present application, “labyrinthulomycetes” is a comprehensive term that includes microorganisms of the orders Thraustochytrid and Labyrinthulid, and includes (without limitation) the genera Althornia, Aplanochytrium, Aurantiochytrium, Botyrochytrium, Corallochytrium, Diplophryids, Diplophrys, Elina, Japonochytrium, Labyrinthula, Labryinthuloides, Oblongichytrium, Pyrrhosorus, Schizochytrium, Thraustochytrium, and Ulkenia. Examples of suitable microbial species within the genera include, but are not limited to: any Schizochytrium species, including, but not limited to, Schizochytrium aggregatum, Schizochytrium limacinum, Schizochytrium minutum, Schizochytrium mangrovei, Schizochytrium marinum, Schizochytrium octosporum, and any Aurantiochytrium species, any Thraustochytrium species (including former Ulkenia species such as U. visurgensis, U. amoeboida, U. sarkariana, U. profunda, U. radiata, U. minuta and Ulkenia sp. BP-5601), and including Thraustochytrium striatum, Thraustochytrium aureum, Thraustochytrium roseum; and any Japonochytrium species.

In some embodiments, the genome is a bacterial genome, an archaea genome, a yeast genome, an algae (e.g., a single-cell algae) genome, a fungi (e.g., a single-cell fungi) genome, or a protozoa genome. Examples of bacterial genome include, but are not limited to, genome of gram positive bacteria, genome of gram negative bacteria. In some embodiments, the genome is a genome of Mycoplasma genitalia (M. genitalium), genome of M. mycoides, genome of M. capricolumn (e.g., subspecies capricolum), genome of E. coli, genome of B. subtilis, or a combination thereof. The genome can also vary in the number of chromosome. For example, the genome may only have a single chromosome or multiple chromosomes (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more chromosomes). In some embodiments, the genome is a single chromosome genome. In some embodiments, the genome is a multi-chromosome genome. In some embodiments, the genome is a genome of S. cerevisiae, a genome of K. marxianus, or a combination thereof. In some embodiments, the genome has three to ten chromosomes. In some embodiments, the genome has eight chromosomes.

As used herein, the term “algae” includes cyanobacteria (Cyanophyceae), green algae (Chlorophyceae), yellow-green algae (Xanthophyceae), golden algae (Chrysophyceae), brown algae (Phaeophyceae), red algae (Rhodophyceae), diatoms (Bacillariophyceae), and “pica-plankton” (Prasinophyceae and Eustigmatophyceae). Also included in the term “algae” are members of the taxonomic classes Dinophyceae, Cryptophyceae, Euglenophyceae, Glaucophyceae, and Prymnesiophyceae. Microalgae are unicellular or colonial algae that can be seen as single organisms only with the aid of a microscope. Microalgae include both eukaryotic and prokaryotic algae (e.g., cyanobacteria). Photosynthetic bacteria include cyanobacteria, green sulfur bacteria, purple sulfur bacteria, purple nonsulfur bacteria, and green nonsulfur bacteria. Examples of genomes suitable for use in the methods disclosed herein include, but are not limited to, Achnanthes, Amphiprora, Amphora, Ankistrodesmus, Asteromonas, Boekelovia, Borodinella, Botryococcus, Bracteococcus, Chaetoceros, Carteria, Chlamydomonas, Chlorococcum, Chlorogonium, Chlorella, Chroomonas, Chrysosphaera, Cricosphaera, Crypthecodinium, Cryptomonas, Cyclotella, Dunaliella, Ellipsoidon, Emiliania, Eremosphaera, Ernodesmius, Euglena, Franceia, Fragilaria, Gloeothamnion, Haematococcus, Halocafeteria, Hymenomonas, Isochrysis, Lepocinclis, Micractinium, Monoraphidium, Nannochloris, Nannochloropsis, Navicula, Neochloris, Nephrochloris, Nephroselmis, Nitzschia, Ochromonas, Oedogonium, Oocystis, Ostreococcus, Pavlova, Parachlorella, Pascheria, Phaeodactylum, Phagus, Platymonas, Pkurochrysis, Pleurococcus, Prototheca, Pseudochlorella, Pyramimonas, Pyrobotrys, Scenedesmus, Schizochytrium, Skeletonema, Spyrogyra, Stichococcus, Tetraselmis, Thraustochytrium, Thalassiosira, Viridiella, and Volvox species. Photosynthetic bacteria include, for example, green sulfur bacteria, purple sulfur bacteria, green nonsulfur bacteria, purple nonsulfur bacteria, and cyanobacteria. Cyanobacterial species include, without limitation, Agmenellum, Anabaena, Anabaenopsis, Anacystis, Aphanizomenon, Arthrospira, Asterocapsa, Borzia, Calothrix, Chamaesiphon, Chlorogloeopsis, Chroococcidiopsis, Chroococcus, Crinalium, Cyanobacterium, Cyanobium, Cyanocystis, Cyanospira, Cyanothece, Cylindrospermopsis, Cylindrospermum, Dactylococcopsis, Dermocarpella, Fischerella, Fremyella, Geitleria, Geitlerinema, Gloeobacter, Gloeocapsa, Gloeothece, Halospirulina, Iyengariella, Leptolyngbya, Limnothrix, Lyngbya, Microcoleus, Microcystis, Myxosarcina, Nodularia, Nostoc, Nostochopsis, Oscillatoria, Phormidium, Planktothrix, Pieurocapsa, Prochlorococcus, Prochloron, Prochlorothrix, Pseudanabaena, Rivularia, Schizothrix, Scytonema, Spirulina, Stanieria, Starria, Stigonema, Symploca, Synechococcus, Synechocystis, Tolypothrix, Trichodesmium, Tychonema, and Xenococcus species.

The size of the genome (e.g., the first genome, the second genome, the third genome, and the synthetic genome of interest) can vary. For example, the genome can be, be about, be at least, or be at least about, 10 kilobase (kb) to about 200 megabase (Mb) in length. In some embodiments, the genome is, or is about, 10 kb, 50 kb, 100 kb, 150 kb, 200 kb, 250 kb, 300 kb, 350 kb, 400 kb, 450 kb, 500 kb, 550 kb, 600 kb, 650 kb, 700 kb, 750 kb, 800 kb, 850 kb, 900 kb, 950 kb, 1 Mb, 1.1 Mb, 1.2 Mb, 1.3 Mb, 1.4 Mb, 1.5 Mb, 1.6 Mb, 1.7 Mb, 1.8 Mb, 1.9 Mb, 2 Mb, 2.1 Mb, 2.2 Mb, 2.3 Mb, 2.4 Mb, 2.5 Mb, 2.6 Mb, 2.7 Mb, 2.8 Mb, 2.9 Mb, 3 Mb, 3.1 Mb, 3.2 Mb, 3.3 Mb, 3.4 Mb, 3.5 Mb, 3.6 Mb, 3.7 Mb, 3.8 Mb, 3.9 Mb, 4 Mb, 4.5 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 15 Mb, 20 Mb, 30 Mb, 40 Mb, 50 Mb, 100 Mb, 200 Mb in length, or a range of any two of these values (including the end points). In some embodiments, the genome is at least, or is at least about, 10 kb, 50 kb, 100 kb, 150 kb, 200 kb, 250 kb, 300 kb, 350 kb, 400 kb, 450 kb, 500 kb, 550 kb, 600 kb, 650 kb, 700 kb, 750 kb, 800 kb, 850 kb, 900 kb, 950 kb, 1 Mb, 1.1 Mb, 1.2 Mb, 1.3 Mb, 1.4 Mb, 1.5 Mb, 1.6 Mb, 1.7 Mb, 1.8 Mb, 1.9 Mb, 2 Mb, 2.1 Mb, 2.2 Mb, 2.3 Mb, 2.4 Mb, 2.5 Mb, 2.6 Mb, 2.7 Mb, 2.8 Mb, 2.9 Mb, 3 Mb, 3.1 Mb, 3.2 Mb, 3.3 Mb, 3.4 Mb, 3.5 Mb, 3.6 Mb, 3.7 Mb, 3.8 Mb, 3.9 Mb, 4 Mb, 4.5 Mb, 5 Mb, 6 Mb, 7 Mb, 8 Mb, 9 Mb, 10 Mb, 15 Mb, 20 Mb, 30 Mb, 40 Mb, 50 Mb, 100 Mb, or 200 Mb in length. In some embodiments, the genome size is no more than 5 Mb, no more than 8 Mb, no more than 10 Mb, no more than 12 Mb, no more than 15 Mb, no more than 18 Mb, or no more than 20 Mb. In some embodiments, the genome size is about 3 Mb to about 13 Mb.

The methods described herein can comprise testing a genome for one or more properties, for example a set of desired properties. For example, in some embodiments, step (b) comprises testing the second genome for the set of desired properties. In some embodiments, the method comprises a step (e) testing the third genome for the set of desired properties. The genome can be tested for properties such as viability in one or more environments (e.g., in vivo or in vitro chemical or biological systems), growth rate, doubling time, certain metabolism capability, adaptability, or a combination thereof. As described herein, testing a genome (e.g., the second genome or the third genome) for one or more properties can comprise, for example, introducing the genome into a cell or a cell-like system and testing for the properties. The genome can be introduced to the cell or the cell-like system by, for example, conjugation, transformation, transduction, or any combination thereof. In some embodiments, the cell-like system can be, or can comprise, a membrane-bound volume, a lipid vesicle, a cell from which one or more intracellular components have been removed, a cell from which the resident genome has been removed, or any combination thereof. In the method disclosed herein, modifying at least one of the second genome fragments in step (f) is, in some embodiments, at least partly based on the testing of step (e). In some embodiments, modifying at least one fragment of the second genome in step (f) further comprises conducting mutation study of the at least one fragment and modifying the at least one fragment at least partly based on the mutation study.

As disclosed herein, it can be advantageous to use conjugation to transfer synthetic chromosome(s) into K. marxianus cell. In some embodiments, shortly after or during the transfer, the resident (e.g., native) chromosome of the K. marxianus cells can be targeted at multiple locations using CRISPR/Cas9, which can result in a strain that only carries the synthetic genome. The resident (e.g., native) chromosome can, in some embodiments, be lost during propagation, unable to replicate, after multiple double-stranded breaks are introduced by CRISPR/Cas9. Non-limiting examples of methods suitable for use to remove resident (e.g., native) chromosome after introduction of the synthetic genome include URA3-FOA based negative selection of the native chromosome described in Boeke J D, et al. (1987) Methods Enzymol 154:164-75 and onducible-inactivation of the centromere of the native chromosome described in Hill A, Bloom K (1987). Mol Cell Biol. 7(7):2397-405.

The methods described herein can include designing a second genome based on the first genome and the second genome is hypothesized to have a set of desired properties. Information from various sources can be used in modifying nucleic acid sequences, for example genomic fragments. In some embodiments, information from various sources can be used to modify the first genome for designing the second genome, and/or to modify the fragment(s) of the second genome, e.g., before repeating steps (d)-(f) in one or more iterations. For example, information from knowledge known in the art, literature resources, experimental data, or any combination thereof can be used. The literature resources can be, for example, scientific publications (e.g., journal articles, conference posters, online publications). The experimental data can, for example, comprises data obtained from mutation studies of the first genome, a genome related to the first genome, or any combination thereof. The mutation studies can be studies of deletion and/or modification of single or multiple genes or non-coding regions, mutagenesis studies (e.g., targeted or random mutagenesis), studies of deletions and/or modification of non-coding genomic regions, gene knockout studies, and add-back studies. Non-limiting examples of mutagenesis studies include transposon mutagenesis, insertional mutagenesis, site-directed mutagenesis, and single- or multiple-site plasmid mutagenesis. In some embodiments, the experimental data comprises data related to genes of essential function redundancies (EFR), which are also referred to as essential function pairs (EFP). In organisms (e.g., a bacterium), certain essential (or quasi-essential) functions is provided by more than one gene. These genes may or may not be paralogs. Suppose gene A and gene B, each supply an essential function E1. The gene pair (gene A and B) represents an EFP. Either gene (gene A or B) can be deleted without loss of the essential function E1, so each gene by itself in a single knockout study is classified as non-essential. However, if both gene (i.e., genes A and B) are deleted, the cell is not viable because the essential function E1 is no longer provided. In some embodiments, one of the EFP is deleted from the first genome in designing the second genome. In some embodiments, only one of the EFP is kept in the second genome.

In some embodiments, the third genome comprises one or more fragments from a naturally occurring genome and one or more fragments from a synthetic genome.

Synthesizing and Assembling Nucleic Acid Molecules

In some embodiments of the method disclosed herein, the combining step comprises combining each of the fragments of the second genome with fragments of the first genome to generate a plurality of third genomes having all N corresponding fragments. In some embodiments, each of the plurality of third genomes is tested for the set of desired properties. In some embodiments, two or more of the plurality of third genomes is tested for the set of desired properties. In some embodiments, one or more of the plurality of third genomes is not tested for the set of desired properties.

Nucleic acid molecules (e.g., genomic fragments) can be produced by a variety of methods, including but not limited to, genetically engineered, amplified, and/or expressed/generated recombinantly. Techniques for the manipulation of nucleic acid sequences, such as, e.g., subcloning, labeling probes (e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature. In addition, the nucleic acid molecules can be synthesized in vitro, such as by well-known chemical synthesis techniques, and/or obtained from commercial sources, and optionally assembled, such as for large nucleic acids and genomes, for example, as described in US20090275086.

Any methods or techniques suitable for combining genomic fragments may be used herein to combine the fragment(s) of the second genome with fragment(s) of the first genome to generate a third genome having all N corresponding fragments. The fragments of the first and the second genomes can be produced using any methods for suitable nucleic acid synthesis, including but not limited to, chemical synthesis, recombinant production, and any combination thereof. Each of the fragments of one genome does not need to be synthesized or produced using the same method. For example, one fragment of the first genome may be synthesized chemically and the remaining fragments of the first genome may be recombinantly produced. In some embodiments, all fragments of the first gnome are synthesized chemically. In some embodiments, all fragments of the first gnome are synthesized chemically. In some embodiments, at least one fragment of the first gnome is produced recombinantly. In some embodiments, at least one fragment of the second gnome is produced recombinantly. In some embodiments, at least one fragment of the first gnome is synthesized chemically. In some embodiments, at least one fragment of the second gnome is synthesized chemically. In some embodiments, all fragments of the first gnome are produced recombinantly. In some embodiments, all fragments of the second gnome are produced recombinantly. In some embodiments, two or more fragments of the first genome are produced together, for example, and present in the same nucleic acid molecule.

In some embodiments, the combining step comprises chemically synthesizing and assembling the fragments of the first and second genomes to generate the third genome. In some embodiments, assembling the fragments of the first and second genomes comprises assembling chemically synthesized, overlapping oligonucleotides into one or more of nucleic acid cassettes. In some embodiments, the entire synthetic genome of interest is constructed from nucleic acid components that have been chemically synthesized, or that have been created from copies of the chemically synthesized nucleic acid components. Genomic fragments can be constructed using methods know in the art. For example, the genomic fragments can be synthetically constructed using the methods described in US20070264688. In some embodiments, a set of overlapping nucleic acid cassettes are constructed, each generally having about 1.4 kb, 5 kb or 7 kb, which comprise subsets of the genes; and the cassettes are then assembled to form the genomic fragments. The function and/or activity of the genome can be further studied by introducing the assembled genome into a suitable biological system and monitoring one or more functions and/or activities encoded by the genome.

Various methods can be used to generate and assemble nucleic acid cassettes. For example, a cassette of interest can be firstly subdivided into smaller portions from which it may be assembled. In some embodiments, the smaller portions are oligonucleotides of about 30 nucleotides (nt) (e.g., 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, or 32 nt) and about 1 kilobase (kb) in length. In some embodiments, the oligonucleotides about 50 nt (e.g., between about 45 nt and about 55 nt) in length. In some embodiments, the oligonucleotides are designed so that they overlap adjacent oligonucleotides, to facilitate their assembly into cassettes. For example, for M. genitalium, the entire genome sequence may be divided into a list of overlapping 48-mers with 24 nucleotide overlaps between adjacent top and bottom oligonucleotides. The oligonucleotides may be synthesized using conventional methods and apparatus, or they may be obtained from well-known commercial suppliers.

Many methods that can be used to assemble oligonucleotides to form longer molecules, such as cassettes of interest, have been described, e.g., in Stemmer et al. (1995) (Gene 164, 49-53) and Young et al. (2004) (Nucleic Acids Research 32, e59). One non-limiting suitable method, called polymerase cycle assembly (PCA), was used by Smith et al. (2003) (Proc Natl Acad Sci USA 100, 15440-5) for the synthesis of the 5386 nt genome of bioteriophage phiX174. In some embodiments, the cassettes are cloned and/or amplified to generate enough material to manipulate readily. In some embodiments, the cassettes are cloned and amplified by conventional cell-based methods. In some embodiments, e.g., when it is difficult to clone a cassette by conventional cell-based methods, the cassettes are cloned in vitro. One non-limiting example of such in vitro method is described in WO 2006/119066 which uses rolling circle amplification, under conditions in which background synthesis is significantly reduced.

Cassettes which may be generated according to various exemplary methods may be of any suitable size. For example, cassettes may range from about 1 kb to about 20 kb in length. In some embodiments, the cassettes is about 4 to about 7 kb, e.g., about 4.5 to about 6.5 kb, preferably about 5 kb in size. The term “about” with regard to a particular polynucleotide length, as used herein, refers to a polynucleotide that ranges from about 10% smaller than to about 10% greater than the size of the polynucleotide. In order to facilitate the assembly of cassettes, it is preferable that each cassette overlaps the cassettes on either side, e.g., by at least about 50, 80, 100, 150, 200, 250 or 1300 nt. Larger constructs (up to the size of, e.g., a minimal genome) comprising groups of such cassettes are also included, and may be used in a modular fashion according to various exemplary embodiments and methods.

A variety of methods may be used to assemble the cassettes. For example, cassettes may be assembled in vitro, using methods of recombination involving “chew-back” and repair steps, which employ either 3′ or 5′ exonuclease activities, in a single step or in multiple steps. Alternatively, the cassettes may be assembled with an in vitro recombination system that includes enzymes from the Dienocuccus radiodurans homologous recombination system. Methods of in vivo assembly may also be used.

The synthetic genome, for example the third genome, can be further manipulated, either before or after it is identified as the synthetic genome of interest. Non-limiting examples of manipulation include modifying (e.g., deleting, altering individual nucleotides, etc.) one or more of genes in the synthetic genome or deleting entire genes within one or more of the cassettes; replacing genes or cassettes by other genes or cassettes, such as functionally related genes or groups of genes; rearranging the order of the genes or cassettes (e.g., by combinatorial assembly); or a combination thereof. The effects of such manipulations can be examined by re-introducing the synthetic genes into a suitable biological system. Non-limiting factors that can be considered include, e.g., growth rate, nutritional requirements and other metabolic factors.

Any of the genomes disclosed herein, for example the first genome, the second genome, the third genome, and the synthetic genome of interest, can be modified to reorganize gene order. In some embodiments, the order of one or more genes in the genome is changed. In some embodiments of the method disclosed herein, step (d) further comprises reorganizing gene order in the at least one fragment of the second genome before combining it with fragments of the first genome to generate the third genome. In some embodiments, the method disclosed herein further comprises reorganizing gene order in the third genome after it is identified as a synthetic genome of interest. In some embodiments, reorganizing gene order comprises grouping genes related to the same biological process in the at least one fragment of the genome. Non-limiting examples of the same biological process include glucose transport and catabolism; ribosome biogenesis; protein export, DNA repair; transcription; translation; nucleotide synthesis, metabolism and salvage; glycolysis; metabolic processes; proteolysis; membrane transport; rRNA modification; tRNA modification; and any combination thereof.

When the method is used to generate a viable genome of reduced size, in some embodiments, the size of the third genome may be further reduced after the third genome is identified as a viable genome of reduced size. The size of the third genome may be further reduced by, for example, deleting one or more genes from the third genome, deleting one or more non-coding region(s) (e.g., promoter region, enhancer region, and intron region) from the third genome, or a combination thereof. In some embodiments, step (b) comprises testing the second genome for viability. In some embodiments, modifying at least one fragment of the second genome fragments in step (f) further comprises conducting mutation study of the at least one fragment and modifying the at least one fragment at least partly based on the mutation study.

Modularization of Genomes

Also provided herein are methods for modulating genomes. In some instances, it can advantageous to reorganize genes in a genome. For example, genes involved in the related biological processes may not be present in a naturally-occurring genome in adjacent location, and downstream genetic engineering can be more efficient by placing these genes in the same genomic location. In some embodiments, the genes involved in the related biological processes are identified and the orders of these genes are changed to make these genes grouped together in a given location of the genome.

The modularization genomes can be done in a stepwise fashion. For example, a genome of interest can be divided into small fragments (e.g., 32, 64, 72, or more fragments), and orders of the genes in each of the genomic fragments can be changed to form gene clusters. Then, the resulting genome with the first round of gene organization can be divided into larger fragments (e.g., 2, 4 or 8 fragments) for further gene shuffling. The steps can be repeated until a genome with desired gene orders is generated.

Various steps can be performed in the process of modularizing and minimizing a genome, including but are not limited to the following:

(1) Determine essential genes.

(2) Remove non-essential genes and intergenic regions.

(3) Classify essential genes according to function. In some embodiments, genes with related functions (subsystems) will ultimately be represented as contiguous modules of DNA sequence, which can decrease labor and material costs during strain engineering. Multiple change—and hence redirection or optimization of a bug and its subsystems—can be installed by altering the DNA module instead of numerous genes scattered about a genome.

(4) Determine breakpoints between co-located genes that do not impinge on the same function or subsystem. In general, each desired gene must be transcribed for its function to be expressed, and most genes are proteins. These require translation in addition to transcription. Each structural gene (or cotranscribed set of genes) therefore be preferred to be accompanied by intergenic DNA sequences that ensure the gene itself is transcribed and translated as required. In some embodiments, when genes with unrelated functions are physically separated and modules are remade (i.e., co-locating related gene functions), the intergenic regions for co-transcribed genes can be duplicated and/or assigned to one of the genes in the transcription unit. In some embodiments, assignment is preferred over duplication since duplicated sequences can result in genome instability.

(5) The first gene transcribed by a promoter (used by many downstream genes) is assigned to that promoter. In some embodiments, this step includes determining the upstream boundary of the promoter (i.e. intergenic region) that is responsible for transcription and translation of the first gene.

(6) The promoter boundaries are identified and set. Various factors that can be used in the step include, but are not limited to, (6a) the location of terminators can be used. Terminators stop transcription and routinely include an RNA hairpin followed by a T-rich run of 5-20 nucleotides. As they stop transcription, a terminator is unlikely to be located between a gene and its promoter. (6b) Transposon mutagenesis provides a second means for identifying the upstream boundary of a genes promoter. Insertion within a promoter or between a promoter and its gene would likely disrupt the promoters function. In many cases, transposon insertions within or on the gene-proximal side of a promoter are absent or rare. Starting at the upstream boundary the number of insertions can increase dramatically. 6c) Size of the intergenic region, RNAseq data, promoter prediction etc, can all be used to define a probable upstream boundary. And 6d) all of metrics a-c can be assigned scores and scores for different metrics weighted by hand or according to some optimization scheme. These can be used to produce graphical outputs of likely breakpoints on a genome annotated with ORFs, terminators, etc. A person with skill in the art can then make a final decision about breakpoints based on all of this data and its presentation without undue experimentation.

(7) The genome can be fragmented at the breakpoints (e.g., in silico). Fragments that represent a particular subsystem are binned. Thus, for instance, all fragments for glycolysis (i.e. all of the genes encoding the glycolysis) are grouped. All other targeted subsystems are similarly binned.

(8) Some of the genes (or transcription units) that represent a subsystem will have promoters and some of them will not.

(9) Genes without promoters are assigned new promoters from the pool of intergenic regions that were removed during minimization. Alternatively, promoters from other organisms could be used. Several metrics can be employed to match the genes original expression strength and the expression strength of the new promoter. RNAseq data for instance. The point is, such data can be collected, scored, and weighted to identify a likely match. So far, an apparent similarity in the strength of the Shine-Dalgarno sequence (of the new promoter) and the Shine-Dalgarno of the promoterless gene has been sufficient.

(10) After all genes have old or new transcription and translation signals, they are assembled and tested. In some embodiments, a subsection of a genome is modularized and built. Once its function is ensured, the submodules within it can be combined with equivalent submodules from other genome locations to produce a fully modularized organism. Combining submodules into a full module can require no new sequence changes, and only involve changing the relative location of a submodule(s) within a genome.

Many public resources are available for identifying functions of genes and/or enzymes, and for classifying genes and/or enzymes of particular function(s). Non-limiting examples of the public resources include: IUBMB enzume nomenclature (http://www.chem.qmul.ac.uk/iubmb/), Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa and Goto 2000, http://www.genome.jp/kegg/), the GenomeNet (Kanehisa et al. 2002, http://www.genome.jp/), MetaCyc (Caspi et al. 2006, http://metacyc.org/), the Comprehensive Microbial Resource (CMR) (Peterson et al. 2001) at The Institute for Genomic Research (TIGR) (http://www.tigr.org/), the Protein Data Bank (PDB, http://www.rcsb.org/pdb/home/home.do), UniProt (http://www.uniprot.org/), the STRING database (http://string-db.org/), and the PROFESS (protein function evolution structure sequence) database (http://cse.unl.edu/˜profess/). Moreover, the BRENDA enzymatic database (Schomburg et al. 2004, http://www.brenda-enzymes.org/) and ExPASy ENZYME database (Bairoch 2000, http://enzyme.expasy.org/) can be used to, for example, identify substrates and/or products and stoichiometry of reactions catalyzed by individual enzymes and characterize unresolved pathways. The BRENDA database can also be parsed to obtain a list of all enzymes catalyzing irreversible reactions under physiological conditions.

Testing Properties of Genomes

The genomes described herein (e.g., the third genome produced by combining fragments of the first and second genomes) can be introduced to various environments (e.g., biological systems) that allow it to function for testing for its properties (e.g., one or more of the desired properties). For example, the genome may be present in (e.g., introduced into) a suitable biological system allowing proteins, RNAs, DNAs to be produced from the genome.

Prior to being introduced to various environments for testing properties, the genome may be propagated in and/or isolated from cells or tissues. The genome can be isolated from cells or tissues, or can be introduced (for example, conjugated, transformed, transduced, or a combination thereof) into and propagated within other cells, using well-known cloning, cell, and plasmid techniques and systems. The genome sequence in the cells can be natural or synthetic, including partially synthetic. In some cases, the genome sequences may be amplified, such as by PCR, after isolation from cells or tissues. The genome sequence can also be chemically synthesized in vitro using chemical synthesis and assembly methods and, thus, are not isolated from any particular tissue or cell prior to use in the described methods. Methods for chemical synthesis of DNA and RNA and assembly of nucleic acids are known, and include oligonucleotide synthesis, assembly, and polymerase chain reaction (PCR) and other amplification methods (such as, for example, rolling circle amplification, whole genome amplification), such as those described herein and in US20090275086. Synthesis of DNA, for example, can be from DNA (e.g., by PCR) or from RNA, e.g., by reverse transcription. Among the nucleic acids are synthetic genomes. Synthetic genomes can be produced, for example, as described herein and in US20090275086.

A variety of suitable biological systems may be used for testing properties of the genome. For example, the genome can contact a solution comprising a conventional coupled transcription/translation system. In such a system, the genome may be able to replicate itself, or it may be necessary to replenish the nucleic acid, e.g., periodically. In some embodiments, the genome is introduced into a vesicle such that the genome is encapsulated by a protective lipid-based material. For example, the genome can be introduced into a vesicle by contacting the genome, optionally in the presence of desirable cytoplasmic elements such complex organelles (e.g., ribosomes) and/or small molecules, with a lipid composition or with a combination of lipids and other components of functional cell membranes, under conditions in which the lipid components encapsulate the synthetic genome and other optional components to form a synthetic cell. In some embodiments, the genome is contacted with a coupled transcription/translation system and is then packaged into a lipid-based vesicle. In some embodiments, the internal components are encapsulated spontaneously by the lipid materials.

The genome can also be introduced into a recipient cell, such as a bacterial or yeast cell, from which some or all of the resident (original) genome has been removed. For example, the entire resident genome may be removed to form a cell devoid of its functional natural genome and the resident genome may be replaced by the foreign genome. Alternatively, the genome may be introduced into a recipient cell which contains some or all of its resident genome. Following replication of the cell, the resident (original) and the introduced (foreign) genome can segregate, and a progeny cell can form that contains cytoplasmic and other epigenetic elements from the cell, but that contains, as the sole genomic material, the introduced genome. Such a cell is a synthetic cell according to various embodiments and methods, and may, in some embodiments, differ from the recipient cell in certain characteristics, e.g., nucleotide sequence, nucleotide source, or non-nucleotide biochemical components.

Various methods, for example in vitro methods, can be used to introduce a genome (synthetic, natural, or a combination thereof) into a cell. Examples of these methods include, but are not limited to, conjugation, transfection, transduction, transformation, electroporation, lipofection, the use of gene guns, and any combination thereof. In some embodiments, the genome, such as a synthetic genome, is immobilized in agar; and the agar plug is laid on a liposome, which is then inserted into a host cell. In some embodiments, the genome is treated to fold and compress before it is introduced into a cell. Methods for inserting or introducing large nucleic acid molecules, such as bacterial genomes, into a cell are sometimes referred to herein as chromosome transfer, transport, or transplantation.

In some embodiments, the synthetic cell comprises elements from a host cell into which it has been introduced, e.g., the whole or part of the host genome, cytoplasm, ribosomes, and membrane. In some embodiments, the components of a synthetic cell are derived entirely from products encoded by the genes of the synthetic genome and by products generated by those genes. Of course, nutritive, metabolic and other substances as well as physical conditions such as light and heat may be provided externally to facilitate the growth, replication and expression of a synthetic cell.

Various exemplary methods may be readily adapted to computer-mediated and/or automated (e.g., robotic) formats. Many synthetic genomes (including a variety of combinatorial variants of a synthetic genome of interest) may be prepared and/or analyzed simultaneously, using high throughput methods. Automated systems for performing various methods as described herein are included. An automated system permits design of a desired genome from genetic components by selection using a bioinformatics computer system, assembly and construction of numerous genomes and synthetic cells, and automatic analysis of their characteristics, feeding back to suggested design modifications.

Also disclosed are synthetic genomes produced by any one of the methods disclosed herein, and synthetic cells produced by introducing the synthetic genome produced by any one of the methods disclosed herein into a cell or a cell-like system. In some embodiments, the cell-like system is a cell from which a resident genome has been removed.

EXAMPLES

Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure.

Experimental Materials and Methods

The following experimental materials and methods were used for Examples 1-6 described below.

Method for Creating Eight Genomic Segments Flanked by NotI Sites to Facilitate Design and Genome Assembly

Purifying large (˜100 kb) centromeric plasmid DNA from yeast for genome assembly is a lengthy procedure. See, e.g., Mushegian, et al., Proc. Natl. Acad. Sci. USA 93:10268-73 (1996); Hutchison et al., Science 286:2165-9 (1999), the content of which is hereby incorporated by reference. Instead of purifying DNA from yeast, a segmented approach allowed the isolation of DNA from the parental bacterial genome. The Syn1.0ΔREΔIS strain (Table 9) was used as the parental strain to create eight NotI strains (NotI-1 to -8 strains). Eight genomes were modified by the TREC method to engineer two NotI recognition sites (GCGGCCGC) in yeast. The locations of NotI sites were either in an intergenic region or Tn5-defined non-essential gene coding region. NotI sites encompassed approximately ⅛^(th) of the genome (FIG. 2A). Each of the eight ⅛^(th) genome segments (NotI-1 to NotI-8) overlapped adjacent segments by 200 base pair (bp) allowing assembly of the segments into a complete genome in yeast via homologous recombination. A non-essential region (24,916-bp) was not included in the design of the 1^(st) ⅛^(th) genome (between NotI-8 and NotI-1), thus a 200-bp overlapping to the adjacent segment 1 was introduced to the end of segment 8 next to the NotI site (FIG. 2A). To further enhance the efficiency of complete genome assembly, the yeast selection markers MET14, was inserted to the genome of the NotI-6 strain by replacing a non-essential cluster (from MMSYN1_0550 to 0591) (FIG. 2A2A). These engineered genomes were transplanted into M. capricolum recipient cell to produce 8 NotI mycoides strains (NotI-1 to -8 strains). To assemble the mycoides genome, genome DNA was prepared in agarose plugs and digested with restriction enzyme NotI, which generated two fragments, the ⅛^(th) and the ⅞^(th) genome. The ⅛^(th) genome was separated from ⅞^(th) genome by field-inversion gel electrophoresis (FIGE), shown in FIG. 2C. The ⅛^(th) genome was then recovered by electro-elution from the agarose gel (FIG. 2C). To assemble the mycoides genome, 8 purified ⅛^(th) genome segments were co-transformed into yeast spheroplasts and selected on appropriate media. A complete genome assembly was evaluated by multiplex PCR (MPCR) and sized by electrophoresis. In general, the rate of successful complete genome assembly was greater than 50%. Both double markers (HIS3 and MET14) selection and lack of yeast DNA contamination in DNA preparation potentially contributed to the high efficiency of genome assembly.

FIGS. 2A-2C show the method used for creating eight NotI strains. FIG. 2A shows the design of NotI restriction sites around the Syn1DREDIS genome for the creation of 8 mycoides NotI strains. The Syn1DREDIS genome was used as the parental genome to create 8 NotI strains. The genome in each strain was engineered by TREC method in yeast with only two unique NotI restriction sites flanking an approximately ⅛th segment of the genome. For example, two NotI site in the genome of NotI strain 1 were generated at locations of NotI-1U and NotI-1D, indicated by arrows. And in the genome of the NotI strain 2, locations of NotI site were generated at locations of NotI-2U and NotI-2D. Each of the ⅛th segment overlaps adjacent segments by 200 bp for genome assembly. A non-essential region (24,916-bp, from gene MMSYN1_0889 to 0904), flanked by two NotI site (NotI-8D and NotI-1U), was not included in the design (indicated in light grey between NotI-1U and NotI-8D at the top of the figure). Thus, a 200 bp overlapping region, represented by vertical line, was inserted upstream of the NotI site, located at the NotI-8D. One non-essential region (from MMSYN1_0550 to 0591) was replaced with the yeast selection markers MET14 in the genome of the NotI strain 6. This non-essential region is indicated in gray between NotI-5D/NotI-6U and NotI-6D/NotI-7U at the bottom left of the figure. Eight NotI-engineered genomes were transplanted from yeast to M. capricolum recipient cells to produce 8 NotI mycoides strains. FIG. 2B shows that ⅛^(th) genome segments were released from mycoides genomes by restriction enzyme NotI and assembled in yeast. FIG. 2C shows that 8 NotI-digested genomic DNA (from 1 to 8) were subjected to 1% agarose gel electrophoresis to separate ⅞th and ⅛th genome (top). The ⅛th genome segments were recovered by electro-elution from the agarose gel and analyzed in 1% agarose gel electrophoresis (bottom).

Global Tn5 Transposon Mutagenesis

Tn5 Puro Transposon Structure and Sequence.

FIG. 3 shows the structure and sequence of Tn5 puro transposon used for global mutagenesis. DNA was ligated into the Smal site of the multiple cloning site (MCS) in the EZ-Tn5 pMOD2 construction vector (Epicentre) to form the complete transposon sequence. EZ-Tn5 pMOD2 containing the transposon was digested with PshAI (New England Biolabs) and gel purified to obtain the Tn5 puro DNA. Puro is the puromycin resistance marker gene. Ptuf is the tuf promoter. Ter triangle is a bidirectional terminator that is downstream of the gyrA gene of Syn1.0. SqPR and SqPF triangles are primer sites for inverse PCR. 19 bp triangles are the Tn5 inverse termini to which Tn5 transposase binds to form the activated transposome used for generating Tn5 mutagenesis libraries.

Tn5 Mutagenesis Procedure.

To make active transposomes, Tn5 transposase was reacted with Tn5 puro DNA according to Epicentre instructions. Briefly, 2 μl of Tn5 transposon DNA (0.1 μg/μl), 2 μl of glycerol, and 4 μl of Tn5 transposase were mixed together by vortexing, incubated for 30 min at room temperature, and then stored at −20° C.

M. Mycoides cells were grown in SP4 media to pH 6.3-6.8. A 40 ml culture was centrifuged at 10° C. for 15 min at 4700 rpm in a 50 ml tube. The pellet was resuspended in 3 ml of Tris 10 mM, sucrose 0.5 M, pH 6.5 (Buffer S/T). The resuspended cells were centrifuged for 15 min at 4700 rpm at 10° C. The pellet, still in the 50 ml tube, was suspended in 250 μl of 0.1 M CaCl₂ and incubated for 30 min on ice. Transposomes (2 μl) and yeast tRNA (Life Technologies) 10 mg/ml (1 μl) were added and mixed gently with the cells. Two mls of 70% Polyethylene glycol (PEG) 6000 (Sigma) dissolved in S/T buffer was added. The suspension was incubated at room temperature with gentle mixing, and then 20 ml S/T buffer was added and mixed well. Cells were centrifuged at 8° C. for 15 min at 10,000×g. The supernatant was discarded and the tube drained well to remove the PEG. The cells were resuspended in 1 ml of warm SP4 media, incubated for 2 hours at 37° C., and then plated on SP4 agar containing 10 μg/ml of Puromycin (Puro) (Sigma). Colonies were generally visible after 3 to 4 days at 37° C.

Colonies (>40,000 total) from several plates were suspended in 22 ml of SP4 media containing puro10 μg/ml (P0). Fifty μl of P0 cells were diluted into 50 ml SP4 puro media and incubated at 37° C. for 24 hours (P1 passage). P1 cells (3 μl) were diluted into 50 ml of fresh SP4 puro medium and grown for 24 h (P2). Two more serial passages were done to yield P3 and P4 cell cultures.

P4 cells were centrifuged at 4700 rpm for 15 min. The cell pellet was resuspended in 300 μl of 0.1 M NaCl, 20 mM NaEDTA solution (pH 8.0) in a 1.5 ml of Eppendorf tube. SDS 10% (30 μl) and proteinase K 5 μg (USB Corporation) were added and mixed well. The tube was incubated overnight at 37° C. The mixture was extracted with 330 μl of aqueous phenol (Sigma) and then centrifuged at 15,000 RPM for 5 min. The supernatant was transferred to a new 1.5 ml tube and extracted with an equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) (Sigma). After centrifugation the supernatant was transferred to a new 1.5 ml tube. A 1/10 volume of 3 M sodium acetate (Sigma) was added and the DNA was ethanol precipitated. After centrifugation at 8,000×g for 5 min the pellet was dissolved in 200 μl of TE buffer (pH 8.0) (Teknova). One μl of RiboShredder™ RNase Blend (Epicentre) was added and the mixture incubated it at 37° C. for 1 hour. The DNA was then extracted with phenol/chloroform, ethanol precipitated, and the pellet dissolved in 200 μl of TE. P0 DNA was similarly prepared from a sample of P0 cells.

DNA enriched for transposon/genomic DNA junctions was obtained by inverse PCR as follows. P0 or P4 DNA (40 μl) in 0.5 ml of TE was sheared in a Nebulizer (Invitrogen™) at 15 lb/in² for 30 sec to obtain 1.5 to 2 kb fragments of DNA. The DNA was ethanol-precipitated, dissolved in 100 μl TE, diluted with 100 μl of 2×BAL31 nuclease buffer and digested with 1 μl of BAL31 nuclease (New England Biolabs, 1U/μl) for 5 min at room temperature to produce blunt-ended DNA. After phenol extraction and ethanol precipitation, DNA was circularized using quick DNA ligase (New England Biolabs). Ligase was inactivated at 65° C. for 20 min. The library was amplified by PCR using 2.5 μl of SqFP and SqRP primers (10 uM each), 20 μl of ligation reaction, and 25 μl of Phusion® High-Fidelity PCR Master Mix (New England Biolabs). The cycling conditions were one cycle at 98° C. for 30 sec, 29 cycles at 98° C. for 15 sec, 58° C. for 20 sec, 72° C. for 3 min with a final extension for 5 min at 72° C. The PCR product, ranging from ≈0.5 to 1 kb, was purified using phenol/chloroform extraction and ethanol precipitation and dissolved in TE at 40 ng/μl.

Tn5 Mutagenesis to Detect Non-Essential Genes.

FIG. 4 shows the steps in producing a Tn5 global insert library. Step 1, Tn5 transposon containing 19 bp mosaic ends, sequencing primer sites, terminator sequences, and a selectable marker (puromycin-resistance gene) was constructed. Transposase (Epicentre) was bound to 19 bp termini to form active transposome. Step 2, transposome was introduced into Mycoplasma mycoides JCVI-Syn1.0 R-M (minus) strain by polyethylene glycol (PEG) transformation method. Puromycin-resistant colonies were collected and were serially propagated to eliminate slow growers. Library P0 was prepared from DNA isolated from colonies. All viable insertions were represented in the library. Library P4 was prepared from final passage (˜50 doublings). Slow growers were lost. Step 3, genomic DNA was isolated, sheared using a nebulizer, and ligated to circularize fragments. Specific fragments were PCR amplified (inverse PCR), and these fragments were sequenced using MiSeq.

Miseq Sequencing.

Paired-end libraries for next generation sequencing were constructed from template DNA according to the manufacturer's protocol (Nextera XT DNA, Illumina, San Diego, Calif., USA). Briefly this method involved using a transposase loaded with adapter oligonucleotides to simultaneously fragment the input DNA and ligate adapter sequences in a single reaction. The adapter sequences were then used to amplify the DNA in a reduced-cycle PCR reaction. During the PCR reaction, unique index sequences were added to both ends of the DNA to allow for dual-indexed sequencing of the pooled libraries. PCR cleanup was performed using a 0.5:1 ratio of Ampure XP (Beckman Coulter) to PCR reaction. Libraries were normalized following Illumina's instructions for XT bead-based normalization. In preparation for cluster generation and sequencing, equal volumes of each normalized library was combined, diluted 25-fold in hybridization buffer, and heat denatured. The final library pool was sequenced according to standard protocols (MiSeq, Illumina, San Diego, Calif., USA).

Tn5 Sequencing Data Analysis and Gene Classification.

The sequence reads were searched for the 19-bp terminus of the Tn5 transposon followed by a 30-bp exact match to genome DNA sequence. In earlier mapping procedures BLAST was used to locate the insertion sites, but this led to a low background of erroneous site locations. This was discovered while investigating Tn5 insertions occurring in known essential genes. For example, it was found consistent insertions at 3 or 4 points in the 5′-terminal third of the dnaA gene. These spurious insertions disappeared when the requirement was shifted to an exact 30-bp match immediately following the 19-bp Tn5 terminus. The junction point between the Tn5 sequence and the genome sequence was taken as the insertion point. A large number of insertions were found. And there were a number of hot spots for insertion, but only the set of unique insertion coordinates were used.

FIG. 5 shows that genes can be classified into 3 categories based on data from global Tn5 transposon mutagenesis. Genes that were hit frequently by both P0 and P4 insertions were classified as non-essential n-genes. Genes hit primarily by P0 insertions but not P4 insertions were classified as quasi-essential, growth impaired i-genes. Genes that were not hit at all, or were sparsely hit in the terminal 20% of the 3′-end or the first few bases of the 5′-end were classified as essential e-genes. The use of transposon mutagenesis to identify non-essential, quasi-essential, growth impaired genes, and essential genes has been described in Hutchison et al., Science 286:2165-9 (1999), which is hereby incorporated by reference. The first complete gene in the figure is a quasi-essential i-gene. The second gene is an essential e-gene. The third complete gene at bottom of figure is classified as an n-gene. Library P0, black bars; library P4, open bars.

FIG. 6 shows the Syn1.0 gene map with Tn5 P4 insertions. Genes are indicated as black arrows. Fine black marks indicate P4 Tn5 insertions. P4 insertions most clearly identified the n-genes since e-genes and i-genes had no hits or were sparsely hit, respectively. Non-essential genes tended to occur in clusters (white arrows) far more than expected by chance. The white arrows indicate the deletions in the RGD1.0 design. Regions of the map between the white arrows were mainly occupied by e-genes and i-genes.

Reduced Genome Designs RGD1.0, RGD2.0, RGD3.0

Each segment was designed separately by deleting the coding sequences of non-essential n-genes following the design rules:

(1) Contiguous clusters of n-genes were deleted, along with intergenic regions internal to the cluster.

(2) Intergenic regions flanking the cluster were retained.

(3) Parts of n-genes that overlapped an e- or i-gene were retained.

(4) Parts of n-genes that contained a ribosome binding site or promoter for an e- or i-gene were retained.

(5) When two adjacent genes were divergently transcribed, it was assumed that the intergenic region separating them contained promoters for transcription in both directions.

(6) If a deletion resulted in converging transcripts, a bidirectional terminator was inserted if not already present.

RGD1.0 and RGD2.0 Design.

Table 1 gives statistics on the sizes of fragments of the designed segments for RGD1.0 and RGD2.0. Sizes and fractional sizes of the 8 segments are listed in Table 1 for the RGD1.0 design and the RGD2.0 design. The final genome lengths were corrected for the 200 bp overlaps between the segments. Segments 1, 3, 4, and 5 were redesigned in RGD2.0, whereas segments 2, 6, 7, and 8 remain the same for both designs. Genes deleted in the RGD1.0 design are indicated by light grey arrows in FIG. 7. The 26 genes added back to RGD1.0 to yield the RGD2.0 design are listed in Table 2 and shown in FIG. 7.

TABLE 1 Comparison of RGD2.0 design to RGD1.0 design. C D E F A B RGD1 Ratio RGD2 Ratio Segment syn1.0 bp bp C/B bp E/B 1 140,739 75,732 0.54 90,161 0.64 2 120,912 49,888 0.41 49,888 0.41 3 133,208 73,958 0.56 88,059 0.66 4 131,623 82,531 0.63 84,750 0.64 5 101,708 56,501 0.56 61,324 0.60 6 189,357 80,747 0.43 80,747 0.43 7 124,976 54,482 0.44 54,482 0.44 8 137,887 66,717 0.48 66,717 0.48 Total 1,080,410 540,566 N/A 576,527 N/A Overlaps −1,601 −1,601 N/A −1,601 N/A Genome Length 1,078,809 538,965 0.5 574,926 0.53

FIG. 7 shows the M. mycoides JCVI-Syn1.0 genome (1078 kb) displayed using CLC software. Dark gray arrows are protein coding genes and white arrows with vertical lines are RNA genes. White dotted arrows are the 8 segments. Light grey arrows are the regions kept in the RGD2.0 design and black arrows are deleted regions. White arrows indicate regions added back to the RGD1.0 design to produce the RGD2.0 design.

Table 2 shows the 26 genes that were identified for add back to RGD1.0 segments 1, 3, 4, and 5 to yield the new RGD2.0 design. Two methods were used to identify genes for add back: (1) Tn5 mutagenesis of RGD2678. The Tn5 mutagenesis data for RGD2678 is shown for lib P0 (0 g) in column 7 and lib P4 (4 g) in columns 8. Columns 3 and 4 show Tn5 mutagenesis data for Syn1.0 and columns 5 and 6 show data for D5 (Tables 3A-3D). (2) Analysis of the viability of 39 cluster deletions in clone 19 and clone 59. Genes that produced non-viable deletions (nv) are in columns 9 and 10.

TABLE 2 The 26 genes identified for add back to RGD1.0 segments 1, 3, 4, and 5 to yield the new RGD2.0 design (See Table 1). s1_0g s1_4g D5_0g D5_4g RGD2678_0g RGD2678_4g RGD24*678 RGD23*45678 Gene Annotation by hand shear shear shear shear shear shear cl.19 del cl.59 del MMSYN1_0035 conserved hypothetical 91 55 74 53 49 40 nv protein MMSYN1_0036 D-lactate dehydrogenase 98 46 60 53 44 20 nv MMSYN1_0037 malate permease 64 58 54 56 38 21 nv MMSYN1_0038 conserved hypothetical 68 34 53 55 33 27 nv protein MMSYN1_0051 conserved hypothetical 8 2 5 4 0 0 nv protein MMSYN1_0054 AhpC/TSA family protein 21 0 20 6 4 0 nv MMSYN1_0060 putative membrance protein 39 37 62 47 39 3 nv MMSYN1_0077 putative hydrolase of the 31 21 24 22 19 6 nv nv HAD family MMSYN1_0078 putative hydrolase 43 10 41 37 16 16 nv nv alpha/beta MMSYN1_0080 conserved hypothetical 16 8 23 9 14 2 protein MMSYN1_0217 glycerol uptake 75 6 63 52 64 10 nv nv facilitator protein MMSYN1_0218 glycerol kinase 137 49 109 77 5 0 nv nv MMSYN1_0219 glycerol oxydase 121 18 85 26 64 5 nv nv MMSYN1_0232 pantetheine-phosphate 10 10 11 15 6 0 adenylyltransferase MMSYN1_0245 putative membrane protein 119 51 105 78 68 8 MMSYN1_0246 E1-E2 ATPase subfamily, 39 3 25 10 36 5 putative MMSYN1_0251 conserved hypothetical 12 7 9 9 5 6 nv protein MMSYN1_0252 oxidoreductase 38 21 43 44 38 58 nv MMSYN1_0256 Amino acid permease 66 44 52 50 39 0 superfamily MMSYN1_0275 putative lipoprotein 3 0 3 5 2 0 MMSYN1_0332 conserved hypothetical 6 6 9 5 1 2 protein MMSYN1_0338 putative lipoprotein 30 20 13 15 11 1 MMSYN1_0444 endopeptidase O 51 30 19 20 20 0 MMSYN1_0477 conserved hypothetical 11 4 5 10 18 0 protein MMSYN1_0494 putative N- 35 14 23 11 20 2 acetylmannosamine MMSYN1_0504 rsml, 16S rRNA Cm1402 19 2 14 5 9 0

Fixing Segment 5 in Syn2.0.

An assembly of all 8 RGD2.0 segments with genes 0455, 0467-0469 added back to RGD2.0 segment 5 did not yield a viable transplant. WT Syn1.0 segment 5 was substituted for the RGD2.0 version. When this assembly (RGD2.0 segs 1234WT5678) was transplanted, colonies were obtained in 3 days and the doubling time in liquid SP4 culture in one measurement was 144 min. Systematic deletion of gene clusters from the WT segment 5 was then undertaken as shown in FIG. 8. More details are given in, for example, Tables 4A-4B.

FIG. 8 shows the list of genes deleted in the RGD2.0 design of segment 5. To arrive at the final structure of the segment 5 used in JCVI-Syn2.0, scarless (TREC) deletions of cluster 33, 36, and 37 were carried out on WT segment 5. The 2 genes 0487 and 0488 that were between cluster 36 and 37 were replaced with gene 0154, which had been deleted from segment 2, but had converted to strong i-genes in the RGD2678 intermediate assembly. These changes to segment 5 resulted in the viable cell, Syn2.0.

Tn5 Mutagenesis of Syn2.0 and Identification of 37 Non-Essential Genes for Removal in RGD3.0 Design.

Tn5 mutagenesis of Syn2.0 was carried out, and 90 genes were reclassified as potentially non-essential in the new Syn2.0 background. These were sub-divided into 3 groups. The first group contained 26 genes frequently classified as i or e in previous rounds of mutagenesis. The second group contained 27 genes that were classified as i- or borderline i-genes in some of the previous Tn5 studies. The third group contained 37 genes that had previously been classified as non-essential in several iterations of Tn5 mutagenesis involving various genome contexts. To create the new RGD3.0 design these 37 were selected for deletion from Syn2.0 (Table 12).

FIG. 9 shows the three design cycles involved in building Syn3.0. The map shows details of the deleted and added back genes in the various cycles. The starting cell was Syn1.0 (1,078,809 bp). Dark grey arrows indicate the Syn1.0 genes. White dotted arrows indicate the 8 segments with 200 bp overlaps. Design cycle 1: Black arrows indicate deletions in the RGD1.0 design. RGD1.0 was not viable, but a combination Syn1.0 segments 1, 3, 4, 5 and RGD1.0 segments 2, 6, 7, 8 was viable (referred to as RGD2678). Design cycle 2A: The white arrows indicate 26 genes that were added back to RGD1.0 segments 1, 3, 4, 5 in an attempt to enable those segments to give a viable cell in combination with RGD2678. This version was only viable when Syn1.0 segment 5 was substituted for the RGD1.0 add back version. Design cycle 2B: Deletions of genes 454-474 and 483-492 from the Syn1.0 segment 5 yielded a viable RGD2.0 (576,028 bp). This was equivalent to adding back additional genes (light grey arrows) to RGD1.0 segment 5 and deleting genes 487-488 (white arrow with horizontal lines). Not shown is the insertion of gene 154 in place of 487-488. Design cycle 3: The white arrows with vertical lines indicate an additional 37 Syn1.0 mycoplasma genes plus 2 vector genes (bla and lacZ) and the rRNA operon in segment 6 that were deleted from Syn2.0 to produce a viable Syn3.0 cell (531,560 bp).

Tn5 Mutagenesis of Syn3.0.

Tn5 mutagenesis was performed on Syn3.0 to determine which genes had Tn5 insertions after serial passaging (P4). Genes originally classified as quasi-essential made up almost the whole population of P4 cells, since the genes in Syn3.0 were then primarily essential e-genes, or quasi-essential i-genes by the original Syn1.0 classification, and only the latter were able to grow. The most highly represented in-, i-, and ie-genes are shown in Tables 3A-3C. Twelve genes originally classified in Syn1.0 as non-essential also had significant inserts in passage P4 (Table 3D).

Tables 3A-3D show Tn5 mutagenesis of Syn3.0 with genes listed having significant numbers of inserts after four serial passages (P4). Columns 1 and 2 show P0 forward and reverse orientation inserts, columns 3 and 4 are for passage P1, and columns 5 and 6 are for passage P4. Column 7 shows the sum of forward and reverse oriented insertions for P4. Column 8 is the gene name, column 9 is the original Syn1.0 gene classification based on Tn5 mutagenesis, and column 10 is the gene functional annotation. The genes were classified into 4 groups A, B, C, and D, shown in Tables 3A, 3B, 3C, and 3D respectively, based on their original Syn1.0 classifications as in, i, ie, and n, respectively. They were further classified according to the numbers of P4 inserts from small to large.

Tables 3A-3D. Tn5 mutagenesis of Syn3.0 with genes listed showing significant numbers of inserts after four serial passages (P4). The genes shown in Tables 3A, 3B, 3C, and 3D were classified as in-, i-, ie-, and n-genes respectively based on the original Syn1.0 classifications.

TABLE 3A Tn5 mutagenesis of Syn3.0 with genes listed classified as in-genes based on the original Syn1.0 classifications. P4_F + HSW g19P0_F g19P0_R g19P1_F g19P1_R g19P4_F g19P4_R P4_R MMSYN1 130821 Hand annotation 8 8 5 8 1 1 2 _0400 in ThiJ/PfpI family protein 3 1 10 3 2 1 3 _0004 in rsmA, ksgA, 16S rRNA m6 2A1518, m6 2A1519 4 1 3 1 2 1 3 _0376 in conserved hypothetical protein 2 2 7 10 1 2 3 _0401 in peptidase C39 family protein 4 3 5 3 1 2 3 _0504 in rsmI, 16S rRNA Cm1402 1 1 6 6 2 2 4 _0409 in NIF3 family protein 1 2 2 5 2 2 4 _0851 in real? 5 3 12 8 3 2 5 _0416 in conserved hypothetical protein 1 1 5 2 2 3 5 _0113 in glycosyltransferase 0 3 0 11 1 4 5 _0421 in conserved hypothetical protein 6 5 11 7 4 2 6 _0381 in MTA/SAH nucleosidase 4 5 1 8 1 5 6 _0046 in recR 6 2 8 6 5 2 7 _0382 in deoxynucleoside kinase 2 4 14 10 5 3 8 _0326 in conserved hypothetical protein 3 4 11 9 3 5 8 _0495 in ROK family protein 5 3 3 1 6 3 9 _0214 in PAP2 superfamily domain membrane protein 6 1 6 6 5 4 9 _0114 in glycosyltransferase 3 0 6 6 5 4 9 _0852 in real? 7 7 11 11 4 5 9 _0838 in rlmB, 23S rRNA Gm2251 36 25 18 15 4 6 10 _0095 in secA 14 19 27 26 7 4 11 _0127 in HD domain protein 19 16 22 21 6 5 11 _0264 in serine/threonine protein kinase 6 8 13 19 5 6 11 _0517 in rluD 13 18 12 23 3 11 14 _0042 in transcriptional regulator, RpiR family protein 6 8 17 14 5 10 15 _0907 in conserved hypothetical protein 3 9 5 15 2 13 15 _0080 in conserved hypothetical protein 9 7 18 14 12 6 18 _0043 in might be rsmC or rlmF 15 5 16 9 11 8 19 _0005 in real? 9 10 20 22 11 8 19 _0494 in putativeN--acetylmannosamine--6-- phosphate2--epim 22 28 40 45 9 13 22 _0305 in Xaa--Pro peptidase, M24 family 67 72 61 73 10 15 25 _0824 in uvrA 12 16 19 31 11 17 28 _0732 in deoxyribose--phosphate aldolase 55 53 69 55 18 16 34 _0825 in uvrB

TABLE 3B Tn5 mutagenesis of Syn3.0 with genes listed classified as i-genes based on the original Syn1.0 classifications. 52 46 45 23 0 2 2 _0316 i transketolase 7 4 0 0 0 2 2 _0394 i ATP-dependent protease La 4 3 0 5 0 2 2 _0525 i protein MraZ 30 31 11 17 0 2 2 _0799 i glycine hydroxymethyl- transferase 10 11 14 14 0 2 2 _0823 i folC folate synthetase- polyglutamyl folate syntheta 24 21 16 13 0 2 2 _0872 i ychF 17 9 10 10 1 2 3 _0240 i thiI, s4U modification in tRNA with icsS 1 3 6 4 0 3 3 _0777 i conserved hypothetical protein 12 24 23 14 2 2 4 _0887 i cdr 6 15 4 13 1 3 4 _0132 i AAA family ATPase 3 5 12 16 1 3 4 _0239 i conserved hypothetical protein 1 0 8 4 4 2 6 _0814 i UDP-galactopyranose mutase 27 20 25 17 3 3 6 _0414 i RelA/SpoT family protein 3 7 5 10 3 3 6 _0620 i ferric uptake regulator 6 6 23 29 3 5 8 _0479 i conserved hypothetical protein 2 6 3 10 3 5 8 _0817 i conserved hypothetical protein (WhiA) 8 4 14 9 7 3 10 _0142 i protein-(glutamine- N5)methyl transferase, release 7 3 21 11 7 4 11 _0347 i cytidylate kinase 10 20 6 24 1 10 11 _0108 i putative lipoprotein 3 4 15 14 7 5 12 _0404 i recO 2 5 6 12 5 7 12 _0853 i conserved hypothetical protein 15 14 20 17 8 5 13 _0697 i glycosyltransferase 48 40 43 46 6 10 16 _0708 i alkyl phosphonate ABC transporter, substrate- bindin 22 29 44 41 6 10 16 _0878 i amino acid permease 8 13 23 22 15 5 20 _0329 i rluB n23 or rsuA n23 21 15 27 31 14 11 25 _0434 i tRNA: M(5)U-54 methyltransferase 16 18 25 30 2 23 25 _0106 i xseA 13 14 20 29 11 17 28 _0435 i phosphomannose isomerase type I 9 15 26 27 10 18 28 _0216 i hypoxanthine phosphoribosyltransferase 13 13 26 26 17 13 30 _0097 i dna polymerase I, 5′-3′ exonuclease 29 17 40 35 25 17 42 _0876 i amino acid permease 3 2 13 16 23 30 53 _0115 i utp-glucose-1- phosphateuridylyl transferase(udp-g 80 77 83 96 33 28 61 _0228 i pdhD 20 27 40 42 31 31 62 _0433 i copper homeostasis protein 46 43 69 70 30 39 69 _0133 i conserved hypothetical protein 14 10 47 42 42 29 71 _0411 i putative membrane protein 89 76 94 91 46 35 81 _0227 i pdhC 9 2 21 14 53 41 94 _0733 i phosphoglucomutase or phosphomannomutase

TABLE 3C Tn5 mutagenesis of Syn3.0 with genes classified as ie- genes based on the original Syn1.0 classifications. 0 1 3 2 1 1 2 _0789 ie ATP synthase, delta subunit 1 1 1 1 0 2 2 _0067 ie 5S ribosomal RNA 8 20 6 7 0 2 2 _0068 ie 23S ribosomal RNA 0 1 1 3 0 2 2 _0910 ie ribosomal protein L34 0 5 2 14 1 2 3 _0301 ie rimP 0 3 1 6 1 2 3 _0873 ie conserved hypothetical protein 2 2 0 2 0 3 3 _0693 ie conserved, caax amino protease family 0 3 10 4 3 1 4 _0346 ie conserved hypothetical protein 3 2 6 2 3 1 4 _0632 ie conserved hypothetical protein 9 5 15 9 8 2 10 _0481 ie lipoprotein, putative (VlcE) 2 2 12 8 6 5 11 _0726 ie glucosamine-6-hosphate deaminase 8 10 9 27 13 35 48 _0109 ie apurinic endonuclease Apn1

TABLE 3D Tn5 mutagenesis of Syn3.0 with genes listed classified as n-genes based on the original Syn1.0 classifications. 2 7 2 7 1 2 _0874 n 16S rRNA methyltransferase GidB 6 5 13 16 4 0 _0640 n tRNA pseudouridine 2 5 8 9 1 4 _0290 n tRNA pseudouridine synthase B 1 0 2 1 1 4 _0462 n IS1296 transposase protein A 5 5 13 11 5 1 _0601 n putative membrane protein 16 22 22 24 5 3 _0060 n putative membrane protein 3 3 15 11 3 6 _0094 n putative membrane protein 4 6 17 16 3 7 _0692 n 23S rRNA pseudouridine synthase 17 9 19 21 8 6 _0505 n putative lipoprotein 15 13 18 21 12 9 _0306 n hypothetical protein 12 17 31 37 9 15 _0444 n endopeptidase O 29 10 39 10 21 6 _0063 n tRNA--dihydrouridine synthase B

Combinatorial Assembly of Intermediate RGDs

All individual ⅛^(th) RGD1.0 segment, together with a ⅞^(th) JCVI-Syn1.0 genome, generated viable transplants, but the assembled complete RGD1.0 genome did not. To analyze potential synthetic growth defects or lethality among ⅛^(th) RGDs, various intermediate RGD was constructed. Intermediate RGD, consisting of different combinations of ⅛^(th) RGD segments, was assembled in a combinatorial manner in yeast and then tested by transplantation. A number of transplants were obtained after two independent assemblies (Tables 4A-4B). A combination of RGD fragments of an intermediate RGD can be determined by the amplicons' patterns of two MPCRs, demonstrated in Table 4B. Among these intermediate RGDs, the RGD2678 which contained 4 RGD segments (RGD1.0-2, -6, -7, and -8) and 4 WT segments (1, 3, 4, and 5) exhibited an acceptable growth rate. The genome of the RGD2678 was sequenced and subjected to the Tn5 mutagenesis to find any non-essential n-gene(s) might become critical for cell growth in the background of this combination genome. From the second assembly screening, another intermediate RGD, RGD24*678, also exhibited an acceptable growth phenotype. Original MPCR data suggested that the genome contained 5 RGD fragments (RGD1.0-2, -4, -6, -7, and -8), but genome sequence data showed that segment 4 is a “hybrid” segment which approximately first ⅓^(rd) WT segment 4 was substituted by RGD1.0-4 sequence. This might result from a recombination between the RGD1.0-4 and the WT 4 segment during genome assembly in yeast. Instead of using the Tn5 mutagenesis approach, this clone was subjected to deletion targeting 39 gene clusters and single genes which had been removed in the design of RGD1.0-1, -3, -4, and -5 (Table 5).

Tables 4A-4B show generation of intermediate RGD transplants. Table 4A shows a list of RGD and WT segments. Different versions of RGD segments were used in each assembly. Table 4B shows various intermediate RGD transplants isolated from 4 independent assemblies. Assembly 1: (RGD1.0-1, -2, -4, -6, -7, and -8)+(WT 1 to 8). Assembly 2: (RGD1.0-1 to -8)+(WT1, 3, 4, 5). Assembly 3: (RGD2.0-3, -4, and -5)+RGD1.0-2, -6, -7, and -8)+WT1; (RGD2.0-1, -4, and -5)+(RGD1.0-2, -6, -7, and -8)+WT3; (RGD2.0-1, -3, and -5)+(RGD1.0-2, -6, -7, and -8)+WT4; and (RGD2.0-1, -3, and -4)+(RGD1.0-2, -6, -7, and -8)+WT5. Assembly 4: (RGD2.0-4, and -5)+RGD1.0-2, -3s, -6, -7, and -8)+WT1; (RGD2.0-1, -4, and -5)+(RGD1.0-2, -6, -7, and -8)+WT3; (RGD2.0-1 and -5)+(RGD1.0-2, -3s, -6, -7, and -8)+WT4; and (RGD2.0-1 and -4)+(RGD1.0-2, -3s, -6, -7, and -8)+WT5. Assembly 1 and 2 were performed by a combinatorial manner. Assembly 3 and 4 were carried out by a combination of 7 RGD segments along with 1 WT segment. The WT6M was used in Assembly 1; the RGD1.0-6sf-LP was used in Assembly 1 and 2; and the RGD1.0-6P-LP was used for Assembly 3 and 4.

Tables 4A-B. Generation of intermediate RGD transplants. Table 4A shows a list of RGD and WT segments. Table 4B shows various intermediate RGD transplants isolated from 4 independent assemblies.

TABLE 4A A list of RGD and WT segments. ⅛ genome Note RGD segment RGD1.0-1 RGD1.0-2 RGD1.0-3 RGD1.0-3s added back three genes (0217, 0218, and 0219) RGD1.0-4 RGD1.0-5 RGD1.0-6 RGD1.0-6sf self-fixed isolate RGD1.0-6sf-LP self-fixed isolate with the insertion of a landing pad RGD1.0-6P fixed promoter RGD1.0-6P-LP fixed promoter with the insertion of a landing pad RGD1.0-7 RGD1.0-8 RGD2.0-1 reversion RGD2.0-3 reversion RGD2.0-4 reversion RGD2.0-5 reversion WT segment WT 1 WT 2 WT 3 WT 4 WT 5 WT 6 WT 6M The MET14 marker insertion WT 7 WT 8

TABLE 4B Various intermediate RGD transplants isolated from 4 independent assemblies. Assembly clone # semi-RGD colony appears (days)* 1 13 RGD27 3 91 RGD2478 4 105 RGD47 3 110 RGD46 3 124 RGD24678 4 139 RGD478 3 140 RGD2678 3 168 RGD478 3 2 11 RGD12678 6 19 RGD24*678 4 90 RGD25678 4 3 18 RGD2345678 5 32 RGD1235678 6 49 RGD1234678 4 4 48 RGD1234678 4 *The colony seen by the naked eyes in days after transplantation.

Table 5 shows a list of 39 genes and cluster deletions. Thirty-nine single genes and gene clusters that had been deleted in the RGD1.0 design were subjected to individual deletion in the RGD2678 clone 140. Deletion of two clusters (0077-0078 and 0217-0219) did not produce transplants (transplantation column, rows 7 and 11). Deletion of 4 targets (0080, 0331-0332, and 0393, and 0503-0505) produced transplants with a slower growth phenotype (indicated as “S” in column 5).

TABLE 5 List of 39 genes and cluster deletions. gene or cluster segment transplantation colony size 1 0014-0016 Seg1 yes 2 0019-0024 Seg1 yes 3 0035-0038 Seg1 yes S 4 0041 Seg1 yes 5 0050-0060 Seg1 yes S 6 0072-0075 Seg1 yes 7 0077-0078 Seg1 no 8 0080 Seg1 yes S 9 0083-0093 Seg1 yes 10 0204-0212 Seg3 yes 11 0217-0219 Seg3 no 12 0223-0226 Seg3 yes 13 0231-0232 Seg3 yes 14 0236-0237 Seg3 yes 15 0241-0246 Seg3 yes 16 0251-0252 Seg3 yes 17 0255-0256 Seg3 yes 18 0261 Seg3 yes 19 0268-0269 Seg3 yes 20 0272-0277 Seg3 yes 21 0292-0293 Seg3 yes 22 0331-0332 Seg4 yes S 23 0349 Seg4 yes 24 0354 Seg4 yes 25 0357-0358 Seg4 yes 26 0367-0369 Seg4 yes 27 0383-0386 Seg4 yes 28 0393 Seg4 yes S 29 0395-0397 Seg4 yes 30 0417 Seg5 yes 31 0436 Seg5 yes 32 0444 Seg5 yes 33 0454-0474 Seg5 yes 34 0476-0477 Seg5 yes 35 0480 Seg5 yes 36 0483-0486 Seg5 yes 37 0489-0492 Seg5 yes 38 0494-0498 Seg5 yes 39 0503-0505 Seg5 yes S

TABLE 6 Deletions of 39 gene and gene clusters. 39 gene or gene clusters deleted 0014-0016 0019-0024 0035-0038  41 0050-0060 0072-0075 0077-0078  80 0083-0093 0204-0212 0217-0219 0223-0226 0231-0232 0236-0237 0241-0246 0251-0252 0255-0256 261  0268-00269 0272-0277 0292-0293 0331-0332 349 354 0357-0358 0367-0369 0383-0386 393 0395-0397 417 436 444 0454-0474 0476-0477 480 0483-0486 0489-0492 0494-0498 0503-0505

Deletions of 39 Gene Clusters and Single Genes in the RGD24*678 Genome.

The plasmid pCORE6 was used as DNA template to amplify the CORE6 cassette for the deletions. Two rounds of PCR were performed at the gene or gene clusters listed in the Table 6. Yeast transformations were selected on SD URA and correct deletions were screened by PCR to detect insertion junctions. Viability of each deletion was examined by transplantation. Table 6 shows the primers used to amplify the CORE6 cassette for the deletions of 39 gene and gene clusters and to detect insertion junctions.

Data from Both Tn5 Mutagenesis and 39 Individual Deletions.

Data from both Tn5 mutagenesis and 39 individual deletions found that 26 n-genes among these 4 WT segments became i- or e-genes. Thus, the RGD was re-designed by adding back 26 genes to RGD1.0-1, -3, -4, and -5 to produce RGD2.0-1, -3, -4, and -5 segments (Table 10). The new design of RGD2.0 thus consisted of 4 segments of RGD1.0-2, -6, -7, and -8, and 4 segments of RGD2.0-1, -3, -4, and -5. The RGD2.0 was assembled and multiple clones of complete genome assemblies were isolated and tested by genome transplantation. No viable transplant was obtained. To analyze potential synthetic growth defect and lethality among RGD segments, 4 genomes consisted of 7 RGD and 1 WT segments (1, 3, 4, or 5) were assembled and transplanted. Three genomes were able to produce transplants, except the genome assembled from 7 RGD and WT segment 3 (Assembly 3 in Table 4B). Among three transplant clones, clone 49 with 7 RGD and the WT 5 combination had a smallest size of the genome and yet exhibited a better growth rate. In parallel, a similar assembly of 7 RGD and 1 WT segment was performed, except the RGD2.0-3 segment was replaced with the RGD1.0-3s, a modified RGD1.0-3 supplemented with three genes (See the Experimental Materials and Methods section). Transplantation result showed that only one genome, clone 48, containing 7 RGD and WT 5 produced transplant. The size of the clone 48 genome was about 10 kb smaller than that of the clone 49 since the RGD2.0-3 contained 7 more genes than RGD1.0-3s does (Table 4B).

Construction of RGD1.0-3s by Adding a Gene Cluster to RGD1.0-3

The gene cluster (MMSYN1_0217 to 0219) was seamlessly inserted back into its original locus by the TREC-IN method as described in Noskov et al., Biol. Proced. Online. 17, 6 (2015), which is hereby incorporated by reference. It was done by 2 rounds of transformation. The CORE6 cassette was first PCR-amplified. About 0.5 to 1 μg of the purified PCR product was transformed to yeast harboring the semi-RGD genome containing the ⅛^(th) RGD1-3 segment and selected on SD (−)URA plate. After junction PCR screening, a positive clone was subjected to the second round of transformation to insert the cluster (3.6 kb) by a knock-in module, consisting of the 3′ KanMX4 gene, a 50-bp repeat sequence, and the cluster. The 3′ KanMX4 gene was PCR-amplified for 18 cycles using the pFA6a-kanMX4 as template. The second round of PCR was performed for 22 cycles using the first round PCR product as DNA template. The cluster was PCR-amplified using the JCVI-Syn1.0 genome as. The second step was performed by co-transformation of these two PCR products with a 50-bp overlapping sequence (shown in bold). The cluster recombined with the 3′ Kan was inserted into the target locus by selection of restoration of kanamycin resistance. After transformation, cells were incubated at YEPD liquid medium overnight 30° C., followed by growing on YEPD plates, supplemented with Geneticin G418. Precise integration was screened by PCR. The procedure of recycling the cassette to produce a scarless insertion was identical to that of the TREC cassette described in the TREC method. The viability of the modified genome was tested by transplantation.

Yeast Strains, Growth Condition, and Genetic Engineering

The yeast Saccharomyces cerevisiae (S. cerevisiae) strains used were VL6-48 (MAT α, his3Δ200, trp1Δ1, ura3-52, lys2, ade2-101, met14) containing the JCVI Syn1.0 or Syn1/ΔREΔIS genome, and VL6-41X. 8 (MATa, his3Δ200, trp1Δ1, ura3-52, lys2, ade2-101, met14). The JCVI Syn1.0 genome has been described in Gibson et al., Science 329:52-56. (2010), which is hereby incorporated by reference. The Syn1/ΔREΔIS genome has been described in Karas et al., Nature methods 10:410-412 (2013), which is hereby incorporated by reference. Yeast cells were grown in standard rich medium containing glucose (YEPD) or galactose (YEPG); or in synthetic defined (SD) minimal dropout medium. Yeast growth media have been described in Noskov et al., Nucleic Acids Res. 38:2570-2576 (2010), which is hereby incorporated by reference. For the URA3/5-FOA counter-selection, cells were grown on SD (−) HIS, supplemented with 5-fluoroorotic acid (5-FOA) (1 mg/ml). URA3/5-FOA counter-selection has been described in Boeke et al., Molecular & general genetics: MGG 197:345-346 (1984), which is hereby incorporated by reference. For kanamycin selection, cells were grown on YEPD, supplemented with 200 μg/ml of Geneticin G418 (Cat #: 11811-023, Life technologies). Yeast transformation was carried out by either lithium-acetate or spheroplast methods. Yeast transformation using the Lithium-acetate method has been described in Gietz et al., Nucleic Acids Res. 20:1425 (1992), which is hereby incorporated by reference. Yeast transformation using the spheroplast method has been described in Kouprina et al., Nature protocols 3:371-377 (2008), which is hereby incorporated by reference.

A number of genetic tools were used to perform gene(s) deletion and insertion in a mycoides genome cloned in yeast. These included (a) the TREC method for scarless gene cluster deletion in the D-serial genome production and in generation of restriction NotI site in 8 NotI strains, (b) the TREC-IN method for scarless gene knock-in and knock-out, (c) insertion of the MET14 marker or a landing pad cassette, and (d) gene or gene cluster deletion by the TRP1 marker and the CORE6 cassette. All cassettes or markers for yeast transformation were generated by PCR using chimeric primers to introduce additional 40 to 50 bp to ends of PCR products for homologous recombination to upstream and downstream of target site. All primers were purchased from IDT (Integrated DNA Technologies). The Advantage HD Polymerase (Clontech), unless otherwise indicated, was used for polymerase chain reaction (PCR). In general, PCR was performed for 30 cycles. In some cases, if two rounds of PCR were involved, the first round was performed for 18 cycles and the second round was performed for 22 cycles using the first round of PCR product as DNA template. The PCR product was, if needed, purified by the MinElute PCR Purification Kit (Qiagen) according the manufacturer's instruction. Approximately 0.5 to 1 μg of PCR product was used for yeast transformation. Correct integration was screened by junction PCR, unless otherwise indicated, to detect the presence of 2 junctions between an insertion marker (or a cassette), and upstream and downstream of target region. In general, at least two positive clones of all engineering mycoides genome were chosen for genome transplantation.

Method for Construction of Single-Segment-RGD Genomes (⅛^(th) RGD+⅞^(th) JCVI Syn1.0)

To aid in testing the functionality of each RGD synthetic segment, semi-RGD genomes consisting of ⅛th RGD piece and ⅞^(th) Syn1.0 genome were constructed and transplanted. A swapping approach, based on recombinase-mediated cassette exchange (RMCE), was developed to improve the efficiency of genome construction. This RMCE approach has been described in Noskov et al., Biol. Proced. Online. 17, 6 (2015), which is hereby incorporated by reference. Eight landing pad yeast strains containing truncated genomes were constructed by replacing individual ⅛^(th) genome segments with a landing pad cassette containing a truncated URA3 gene and MET14 marker flanked by two mutant loxP sites (FIG. 10). These 8 deleted segments were the same regions of the 8 NotI segments described in the Eight-NotI-strains strategy. To build a semi-RGD genome, a landing pad strain was mated with a corresponding opposite mating type yeast strain containing a donor vector with a RGD segment flanked by another two mutant loxP sites. Diploid strains were selected, followed by galactose induction to trigger the exchange of RGD segment with the landing pad via the cre recombinase (FIG. 10). The structure of semi-RGD genome was first verified by PCR analysis for the boundaries between RGD segment and ⅞^(th) Syn1.0 genome. The PCR products were further digested with restriction enzyme NotI to confirm ⅛^(th) RGD segment was flanked with NotI site as design. In general, the successful rate of swapping varied from 10 to 50%. At least two independent yeast clones were chosen for transplantation. All 8 semi-RGD genomes were able to produce colonies on the SP4 medium containing tetracycline and X-gal at 37° C. Generally, colonies could be seen in 3 to 4 days after genome transplantation, except the transplanted cell from the RGD1.0-6+⅞^(th) WT genome which required about 10 days to produce a similar colony size as the others. Several faster growing cells were later isolated from the RGD1-6 transplant. All ⅛^(th) RGD segments can be released by NotI restriction enzyme and purified for genome assembly.

FIG. 10 shows the construction of the ⅛^(th) RGD+⅞ wild type genome by recombinase-mediated cassette exchange (RMCE). Step 1, an approximate ⅛^(th) genome (colored in gray for WT Syn1.0) of the M. mycoides JCVI-Syn1.0 cloned in yeast was replaced with a landing pad cassette flanked by two 34-bp hetero-specific lox sites. Step 2, a donor plasmid containing a corresponding ⅛^(th) RGD, flanked by another two hetero-specific lox sites was introduced into the landing pad strain by yeast mating. Step 3, the ⅛^(th) RGD was exchanged with the landing pad locus via the Cre-mediated recombination. An intron-containing URA3 gene was split into the landing pad and the donor plasmid so that the cassette exchange can be selected by restoration of uracil prototrophy. Step 4, the ⅛^(th) RGD+⅞^(th) WT genome was transplanted to M. Capricolum recipient cells to produce a mycoides strain. Step 5, a ⅛^(th) RGD fragment was purified by NotI digestion and then used for genome assembly.

Construction of Single-Reduced Segment Genomes by RMCE

To build a single RGD segment genome, ⅛^(th) RGD and ⅞^(th) JCVI Syn1.0 were brought together in a diploid strain. A landing pad strain containing the MET14 marker was first crossed with a corresponding opposite mating type yeast strain that contained an ⅛^(th) RGD segment carried by a donor vector with the TRP1 marker. It was done by mixing two opposite strains on yeast extract peptone dextrose (YEPD) plate and incubating the plate overnight at 30° C. Diploid strains were selected on SD (−) TRP (−) MET plate. The expression of Cre recombinase was induced by growing diploid cells on YEP-galactose plate for two days. The event of cassette exchange was screened by the restoration of uracil prototrophy. The cells from the YEPG were replica plated on SD minus uracil [(−) URA] for 2 to 3 days until colonies appeared. The structure of the semi-RGD genome was first verified by PCR analysis for the boundaries between the RGD segment and the JCVI Syn1.0 genome. The PCR products were further digested with restriction enzyme NotI to confirm ⅛^(th) RGD segment was flanked with NotI site as design. Multiple positive clones were chosen for transplantation. Generally, colonies could be seen in 3 to 4 days after genome transplantation, except transplanted cells from the RGD1.0-6 semi-genome which required about 10 days to give a colony similar in size to the others. All ⅛^(th) RGD segments in semi-genomes could be released by NotI restriction enzyme and purified for genome assembly.

The one exception, segment 6, initially was detected as a very small colony which after 10 days produced faster growing sectors as shown in FIGS. 11A-11G. The sequence of several independent clonal isolates revealed mutations that reduced the stability of a stem-loop transcription terminator downstream of the tRNA-His gene. This allowed read through and expression of the essential gene 0621. FIG. 12 shows the sequence of this region in Syn1.0 and as designed in RGD1.0 segment 6. FIGS. 13 and 14 show the various mutations that restored expression of gene 0621 and yielded a viable segment 6.

Engineering Restriction Nod Sites into the Genome

Generation of a NotI site was performed by the TREC method. To engineer a NotI site, 5 to 9 nucleotides were embedded into the primers used for PCR-amplifying the CORE cassette. If a NotI site was generated within a coding region, a reading frame was maintained by inserting either 9 bp or 6 bp to generate a NotI site together with adjacent nucleotides. The production of the CORE cassette was generated by two rounds of PCR. Since a non-essential region (24.9 kb) between the ⅛^(th) segments 1 and 8 was not included in the 8-NotI-segments design (FIG. 2A2A), 208 bp which contained 8 bp of NotI site and a 200 bp overlapping to the adjacent segment 1 was inserted into the location of the NotI-8D. Using the JCVI1.0-Syn1.0 genome DNA as template, a 305-bp fragment was PCR-amplified by a pair of chimeric primers. The 50-bp of the 5′ PCR product was for homologous recombination to the target site and the 50-bp of the 3′ PCR product was overlapped with the 5′ end of a CORE cassette amplified by another chimeric primers. Approximately 0.5 μg of two purified PCR products were co-transformed to yeast. Correct insertion was screened by junction PCR. The procedure of recycling of the CORE cassette was same as that in the D serial strains.

Primers were designed to amplify the cassette for engineering NotI sites, to detect junctions of the CORE3 cassette insertion, and the cassette recycling (pop out). 208 bp was inserted into the location of NotI (FIG. 2A). The CORE cassette for engineering NotI site at the NotI-8D location was PCR-amplified by only 1 round (30 cycles) and the 305 bp containing the 208 bp was also amplified by 1 round (30 cycles). 5 to 9 nucleotides were embedded into primers for the NotI site engineering.

Insertion of MET14 into WT Segment 6

The yeast MET14 marker was PCR-amplified using Yeast W303 genome DNA as template and the Ex Taq DNA Polymerase (TaKaRa), according to the manufacturer's instructions. The PCR product was transformed to the NotI strain 6 and selected on SD (−) without methionine (MET) plate. The correct MET14 marker replacement was screened by colony PCR.

Constructions of 8 Landing Pad Strains

PCR amplification was used to produce a unique landing pad cassette (2,250 bp) that allowed individual replacement of each ⅛th segment into the JCVI Syn1.0 genome by homologous recombination in yeast. Seven landing pad cassettes were PCR-amplified using a plasmid pRC72 as the DNA template to replace segments from 2 to 8. One landing pad cassette was PCR-amplified using a plasmid pRC73 as a DNA template to replace segment 1, which contained a yeast centromeric plasmid (YCp). After transformation, cells were selected on SD (−) MET, transformants with correct replacement of the cassette were screened by junction PCR. The landing pad insertion genome was further confirmed by contour-clamped homogeneous electric field CHEF gel electrophoresis.

Modification of RGD Segment 6 by an Insertion of a Landing Pad

Two versions of RGD segment 6 (RGD1.0-6sf and RGD1.0-6P) were further modified by inserting a special landing pad cassette to produce the RGD1.0-6sf-LP and RGD1.0-6P-LP, respectively. The first one was the mycoides strain that spontaneously self-fixed the expression of gene MMSYN1_0621 and the second was a redesigned segment 6 that contained a corrected promotor for gene 0621. A modified landing pad was generated by 2 rounds of PCR. The first round PCR was performed using a mycoides genome with the insertion of the modified landing pad as DNA template. The second PCR was carried out using the first round PCR product as template. The PCR product was transformed into yeast containing a single-RGD segment genome with either the RGD1.0-6sf or RGD1.0-6P. The cassette was inserted between genes 0601 and 0606 in RGD segment 6. A correct insertion was detected by screening the junctions. Two modified RGD genomes were transplanted to produce cells containing either RGD1.0-6sf-LP or RGD1.0-6P-LP.

Purification of ⅛^(th) Genome Segments

All ⅛^(th) genome segments were isolated from mycoides genomes. The medium and growth condition of growing mycoides cells were described previously in Lartigue et al., Science 317:632-638 (2007), which is hereby incorporated by reference. Briefly, cells were grown in 10 ml SP4 medium containing tetracycline-resistance (tetM) overnight at 37° C. Cells were harvested by centrifugation at 4,575 g for 15 min at 10° C. Cell pellets were washed with 5 ml of washing buffer (10 mM Tris and 0.5 M sucrose, pH 6.5) and harvested again as before. Cells were re-suspended in 100 μl of washing buffer and incubated at 50° C. for 5 min. Cell suspensions were mixed with 120 μl of 2% agarose (Cat #: 16500-500, Invitrogen) in 1×TAE buffer (40 mM Tris-acetate and 1 mM EDTA), pre-warmed at 50° C. Approximately 100 μl of this mixture was added to agarose plug molds (Cat #170-3713 Bio-Rad). After setting 30 min at room temperature, plugs were lysed and proteins were digested with proteinase-K solution [500 μl of the proteinase-K buffer (Bio-Rad) and 20 μl of Proteainase K (>600 mAU/ml) (Cat #19133, Qiagen)] at 50° C. overnight. Agarose plugs were washed once with 1 ml of 0.1× Wash Buffer (Bio-Rad CHEF Genomic DNA Plug Kit) for 30 to 60 min, followed by washing the same buffer containing 0.5 mM PMSF (phenylmethylsulfonyl fluoride) for 30 min. Plugs were equilibrated with 1 ml of 1× Buffer 3 (NEB) for 30 min twice. The plug was treated with 50 units of NotI for 5 hours at 37° C. in 250 μl 1× buffer 3 containing bovine serum albumin BSA (NEB) in 100 μg/ml concentration. After digestion, the plug was loaded on a 1% agarose gel and subjected to Field-Inversion Gel Electrophoresis (FIGE, BioRad, catalog #161-3016) in 1×TAE buffer. The parameters of the electrophoresis were forward 90 V, initial switch 0.1 sec, final switch 10 sec, with linear ramp, and reverse 60 V, initial switch 0.1 sec, final switch 10 sec, with linear ramp for 16 hours at room temperature. Gel was then stained with 1×TAE buffer containing ethidium bromide (0.5 μg/ml) for 30 minutes. To elute ⅛^(th) genome segments, a REC0-CHIP membrane filter (Takara, code #9039) was inserted next to the band. The gel was re-orientated by 90° so that the DNA could be run into the membrane. The gel was subjected to electrophoresis for 2 hours at 3.5 Volt/cm. The RECO-CHIP filter was placed into the RECO-CHIP DNA collection tube and spun for 2 min at 500×g. In general, about 20 to 30 μl DNA solution was collected and kept at 4° C. 1 to 2 μl of purified DNA was analyzed on a 1% agarose gel by FIGE using the same conditions.

Genome Assembly by the Yeast Method

All ⅛^(th) segments of RGD and WT segments used for genome assembly are listed in the Table 4A. Genome assembly was carried out in the yeast VL6-48 strain. The spheroplast transformation procedure has been described previously in Gibson et al., Science 329:52-56. (2010), which is hereby incorporated by reference, with some modifications. Yeast culturea were harvested at an OD₆₀₀≈2. After centrifugation, cell pellets were re-suspended in 20 ml of 1M sorbitol solution and kept at 4° C. for 4 to 20 hours. Approximately 20 to 50 ng of each of the ⅛^(th) genome segments were mixed in a final volume of 50 μl and then added to about 1×10⁸ yeast spheroplasts. After transformation, yeast spheroplasts were regenerated and selected on a sorbitol-containing SD (synthetic defined) medium with appropriate amino acids missing, either (1) SD-HIS, (2) SD-HIS-TRP, or (3) SD-HIS-TRP-MET.

Yeast DNA Preparation for PCR

Yeast cells were patched to an appropriate selection medium and grown overnight at 30° C. Cells (visible amount equal to approximately 1 μl of cell mass) were then picked by pipette tip and twirled in 0.5 ml of a PCR tube containing 10 μl of the zymolyase solution (10 μl of sterile water+0.5 μl of 10 mg/ml of zymolyase 20T (ICN Biochemicals)). The tube was incubated at 37° C. for 1 hour, followed by 15 min incubation at 98° C. 1 μl of zymolyase-treated cells was analyzed by PCR in 10 μl reaction volume using the QIAGEN Fast Cycling PCR Kit (cat #203741, Qiagen), according to the manufacturer's instructions. About ⅓^(rd) of the PCR product was analyzed on a 2% E-gel (Invitrogen) using the E-Gel Power Base Version 4 (Cat #G6200-04, Invitrogen) for 30 min.

Multiplex PCR Screening to Confirm Genome Assemblies

To screen for complete genome assembly, multiplex PCR was performed by QIAGEN Multiplex PCR Kit (cat #206143, Qiagen) using a unique set of primer mixes, each of which contained 8 primer pairs, with the expected amplicon sizes listed in Table 7. DNA prepared from yeast for PCR was described above. In a 15 μl PCR reaction, it contained 1 μl of zymolase-treated yeast cell suspension, 1.5 μl of 10× primer mix, 6 μl of PCR-grade water, and 7.5 μl of the 2× master mix. The PCR conditions were 94° C. for 15 min, then 35 cycles of 94° C. for 30 s, 52° C. for 90 s, and 68° C. for 2 min, followed by 5 min at 68° C. for one cycle. 5 μl of PCR product was analyzed on a 2% E-gel for 30 min.

Combinatorial genome assembly was performed. Each MPCR primer set contained 8 primer pairs to produce amplicons representing each ⅛th segment (WT or RGD). The Set 9 (WT) produced 8 amplicons only from WT segments and the Set 9 (RGD) only produced 8 amplicons from RGD1.0 segments. The Set 10 primer mix can produce 8 amplicons from both WT and RGD. Similarly, the Set 15 and 16 can detect specifically for WT and RGD2.0 segments, respectively in assembly 3 and 4 (See Table 4B).

TABLE 7 Expected Amplicon Size. MPCR set Amplicons (bp) Set 9 (WT) 121 204 256 301 408 515 612 724 Set 9 (RGD) 129 186 257 306 400 486 618 724 Set 10 108 176 266 323 412 513 619 741 Set 15 129 186 268 314 400 486 618 724 Set 16 129 220 258 301 408 515 612 718 Bacterial Strains and Growth Conditions

M. mycoides strain JCVI-Syn1.0 and strains with altered genomes were grown in SP-4 liquid medium supplemented with 17% fetal bovine serum (FBS) or SP-4 solid medium supplemented with 17% either FBS, 1% agar and 150 mg/L X-gal as described previously in Lartigue et al., Science 317:632-638 (2007), which is hereby incorporated by reference. Syn1.0 genome has been described in Gibson et al., Science 329:52-6 (2010), which is hereby incorporated by reference. In some cases, FBS was replaced with same percentage of horse serum (Catalog #: 26050-088, Life technologies) to enhance cell growth.

Genomic DNA Preparation and Transplantation

Total DNA, including mycoides genomic DNA from yeast was prepared in agarose plugs using a CHEF Mammalian Genomic DNA Plug Kit (Bio-Rad), according to the manufacturer's instructions with some modifications. Two agarose plugs were prepared from 30 ml of yeast culture (OD₆₀₀=1.5 to 2.5). Plugs were incubated in 400 μl of lytic buffer supplemented with 2 mg of zymolyase 20T (US Biological) at 37° C. for 2 hrs. The procedure of genome transplantation has been described previously in Lartigue et al., Science 325:1693-1696 (2009), which is hereby incorporated by reference, except that the M. capricolum RE(−) recipient cells were grown at 30° C.

The TREC-IN Method

FIG. 15 shows a diagram of the TREC-IN method. The CORE6 cassette, consisted of the CORE cassette and a 5′ truncated KanMX4 gene, was produced by PCR amplification. Step 1, the PCR product was transformed and selected on SD (−) URA. Correct insertion was verified by junction PCR (L and R). Step 2, two PCR products, 3′ KanMX4 truncated gene and a gene to be knocked in (represented by YFG), were generated and co-transformed into the yeast strain. Two PCR fragments were recombined via an identical sequence (“U” block) after transformation. A 250 bp overlapping sequence (indicated as black bars above 5′Kan and 3′Kan) between 5′ and 3′ KanMX4 gene would recombine to join 2 PCR DNA fragments to integrate to the target site. Transformation was selected on G418 (Geneticin) for the restoration of KanMX4 gene and a correct integration was verified by junction (R1) PCR. Step 3, the identical procedure for removal of the cassette described in FIG. 23 generated a scarless gene insertion confirmed by junction (L1) PCR.

Synthesis and Assembly of Reduced Genomes

Oligonucleotide Design Software.

The oligonucleotide design software searched for a combination of parameters including dsDNA fragment overlap length, number of assembly stages, maximum fragment size, maximum number of fragments to be assembled per assembly stage, and appended vector and restriction site sequences that will yield overlapping oligonucleotides that do not exceed 80 bases in length. An oligonucleotide overlap size equal to half the oligonucleotide length was used. And dsDNA fragment overlap size of 40-80 bp with the exception of the eighth molecules which contained defined 200-bp overlaps was used. At each stage of assembly, a unique set of vector sequences (30 bp) and restriction sites (8 bp) were appended to the 5′ and 3′ ends of each resulting fragment while ensuring the restriction sites were unique to each sequence. These appended sequences facilitated vector assembly for cloning or PCR amplification and insert release (i.e. removal of vector sequences) to expose overlaps for subsequent assembly stages.

Oligonucleotide Pooling.

Oligonucleotides were purchased from Integrated DNA Technologies (IDT), resuspended in TE pH 8.0 to a final concentration of 100 μM, and then pooled and diluted to a per-oligonucleotide concentration of 25 nM. Upon receiving oligonucleotides from IDT in 96 or 384-well plates, plate barcodes were scanned and plates were loaded onto an automated platform (N×p system from Beckman Coulter). A oligonucleotide design manifest file was used to drive the pooling of partitioned oligonucleotides into pools of approximately 50 oligonucleotides per pool, which constituted a single dsDNA fragment. Two styles of automated pooling were leveraged: (1) pooling via a span-8 style pipetting system in which oligonucleotides were re-arrayed in a slower fashion, but with less intervention; and (2) pooling via a 96-well pipetting head in which oligonucleotides were instantly pooled once placed into a partitioned reservoir (high-speed with more intervention).

Single-Reaction Assembly of dsDNA Fragments from Overlapping Oligonucleotides.

Oligonucleotide pools were copied into 96-well PCR plates using Beckman Coulter's N×p system and enzyme master-mixes were dispensed using a bulk reagent dispenser (Preddator from Redd & Whyte). One-step oligonucleotide assembly and amplification reactions were setup using an enzyme-master mix consisting of 1×Q5 (NEB) or 1× Phusion (Thermo Fisher) PCR master mix, 0.04% PEG-8000, 500 nM forward and reverse Stage01 PCR primers, and 2.5 nM of the oligonucleotide pool generated above. In general, PCR cycling parameters were 98° C. for 2 min, then 30 cycles of 98° C. for 30 s and 65° C. for 6 min (increasing 15 sec/cycle), followed by a single 72° C. incubation for 5 min. All thermal-cycling was carried out on Bio-Rad C1000/S1000 cyclers. Products were analyzed on 1% E-gels (Invitrogen) alongside a 1 kb DNA ladder (NEB) (FIG. 16A). In some cases, due to extreme AT content, a 55° C. or 60° C. annealing/extension temperature was used. In general, 48 oligonucleotides of 60-bases in length were combined to generate ˜1.4 kb dsDNA fragments.

Error Correction and Re-Amplification Reactions.

PCR reactions from above were cycled at 98° C. for 2 min, 2° C./s to 85° C., 85° C. for 2 min, 0.1° C./s to 25° C., 25° C. for 2 min, and then stored at 4° C. 2.7 μl template DNA was combined with an 8.3 μl error correction mix containing 5.3 μl water, 2 μl Surveyor mismatch-recognition endonuclease (IDT), and 1 μl Exonuclease III (NEB) diluted 1:4000 in water to 25 units/ml. Reactions were then incubated at 42° C. for 1 hour. Error corrected templates were then PCR amplified in reactions containing 1×Q5 (NEB) or 1× Phusion (Thermo Fisher) PCR master mix, 500 nM forward and reverse Stage01 PCR primers, and 1:50 error corrected DNA template. In general, PCR cycling parameters were 98° C. for 2 min, then 30 cycles of 98° C. for 30 s and 65° C. for 6 min (increasing 15 sec/cycle), followed by a single 72° C. incubation for 5 min. Products were analyzed on 1% E-gels (Invitrogen) alongside a 1 kb DNA ladder (NEB). (FIG. 16B). In some cases, due to extreme AT content, a 55° C. or 60° C. annealing/extension temperature was used to recover synthetic DNA fragments. In general, a 5-10× reduction in error rates was observed compared to untreated samples.

Assembly, Cloning, and MiSeq Sequence Verification of 7-Kb Cassettes.

Equal amounts (˜500 ng) of stage 1 error-corrected PCR fragments were combined 4- or 5-at-a time. One-fifth volume of NotI restriction enzyme (NEB) was added and the reactions were carried out at 37° C. for one hour. The reactions were then processed and concentrated with a PCR cleanup kit (Qiagen). Reactions were then separated on a 1% agarose gel and fragment pools were gel extracted and purified (Qiagen). The overlapping fragments were then simultaneously assembled into a PCR-amplified pCC1BAC cloning vector (REF) using the Gibson Assembly® HiFi 1-step kit (SGI-DNA) and transformed into Epi300 electrocompetent E. coli cells (Illumina) as previously described. Twenty-four colonies from each first stage cassettes were picked from petri plates using an automated colony picking system (QPix system from Molecular Devices). Picking this number of colonies ensured that it was possible to identify error-free cassettes during this initial pass thus reducing the need to recirculate back through the process.

Bacterial colonies were formatted within a deep-well growth block in such a way that dozens of first stage cassettes could be “collapsed” into a single group of 24 wells via Beckman Coulter's NXp (96-well head) after 20 hours of growth in a shaking incubator (FIG. 17). Formatting in this fashion allowed us to screen many clones without having to increase next-generation sequencing libraries construction throughput by 10×. This single grouping of 24 wells was plasmid prepped using Agencourt CosMCPrep (Beckman Coulter) on an automated platform (N×p system from Beckman Coulter), and the resulting plasmid DNA was used to create 24 indexed libraries using Illumina's Nextera XT system per the manufacturer's protocol. Samples were then sequenced on Illumina's MiSeq platform using reagent kit V2 and a 2×150 bp run type. Illumina's Nextera process was also automated by using Eppendorf's epMotion 5073 and Alpaqua's LE Magnet Plate for low volume bead elution.

After MiSeq sequencing was complete, Clone Verification Analysis (CVA) pipeline was then initiated to identify and select error-free cassettes. A manifest file describing a library-reference association matrix was filled out prior to launch of analysis. The sequenced libraries were quality-trimmed using Trimmomatic 0.32. Concurrently, a comprehensive reference sequence was created by inserting the expected cassette sequence at insertion sites of the vector used. The reference indexes were built using bowtie2-build. Mapping of each library was then performed using bowtie2 with default alignment parameters against all appropriate references described in the manifest file.

The variants in the library were detected by analyzing each BAM file using samtools mpileup. The result was then filtered using bcftools' varfilter and saved as a VCF file. Finally, the VCF files were summarized into a single table that would allow quick identification of the error-free cassettes. Furthermore, this output file was used to drive the automated selection of the cultured clones by using Beckman Coulter's NXp platform (span-8).

Assembly of Overlapping DNA Fragments in Yeast.

Error-free cassettes were prepared from 10-ml induced E. coli cultures and inserts were released by digestion with the AsiSI restriction enzyme (NEB). Equal amounts (˜500 ng) were combined as many as 15-at-a time and then processed and concentrated with a PCR cleanup kit (Qiagen). Between 50-250 ng of each fragment were combined with 50 ng EVW vector and transformed into yeast as previously described in Gibson et al., Science 319:1215-1220 (2008), Gibson et al., Science 371:632-638 (2007), Gibson, Curr. Protoc. Mol. Biol. Chapter 3:Unit3.22 (2011), and Gibson et al., Proc. Natl. Acad. Sci. U.S.A. 105:20404-20409 (2008). Yeast clones were first screened by multiplex PCR at the assembly junctions and then by separation of supercoiled DNA on agarose gels alongside a supercoil ladder.

Plasmid DNA Isolation from Yeast.

Yeast centromeric plasmid (YCp) DNA was prepared as follows. The preparation of yeast centromeric plasmic has been described in Gibson et al., Science 371:632-638 (2007) and Gibson, Curr. Protoc. Mol. Biol. Chapter 3:Unit3.22 (2011), which are hereby incorporated by reference. A 5-10 ml S. cerevisiae culture was grown overnight at 30° C. in complete minimal (CM) medium minus tryptophan (Teknova). Cells were centrifuged and resuspended in 250 μl of buffer P1 (Qiagen), containing 5 μl of Zymolyase-100T solution (10 mg/ml zymolyase-100T [US Biological cat. no. Z1004], 50% (w/v) glycerol, 2.5% (w/v) glucose, 50 mM Tris.Cl, pH 7.5). Following an incubation at 37° C. for 1 hour, 250 μl of lysis buffer P2 (Qiagen) were added. Tubes were inverted several times and incubated at room temperature for 5 min. Then, 250 μl cold neutralization buffer P3 (Qiagen) were added, and the tubes were inverted several times, and the samples were microcentrifuged for 10 min at 16,500×g. The supernatant was transferred into a fresh tube and precipitated with 700 μl isopropanol followed by a 70% ethanol wash. The DNA pellet was resuspended in 30-50 μl TE buffer, pH 8.0. To estimate the size of the purified YCp DNA, 10 μl of the plasmid preparation were separated on a 1% agarose gel in 1×TAE buffer (No ethidium bromide) by constant voltage (3 hr at 4.5V cm-1). After the electrophoresis the gel was stained with SYBR Gold and scanned with a Typhoon 9410 imager (GE Healthcare Life Sciences).

Rolling Circle Amplification (RCA) Reactions.

MDA reactions were generally performed using the TempliPhi™ Large Construct DNA Amplification kit (GE Healthcare). Briefly, 4 μl of yeast plasmid preparation were added to 9 μl of sample buffer. The mixture was incubated at 95° C. for 3 min and then placed on ice. Ten microliters of reaction buffer and 0.5 μl of the enzyme were added to the denatured sample mixture. The amplification reactions were incubated at 30° C. for 16-18 hours. The enzyme was inactivated at 65° C. for 10 min. Five microliters of amplified DNA were digested with the restriction enzyme NotI in 50 μl volume at 37° C. for one hour. Twenty microliters of the digest were separated on a 1% agarose gel with EtBr in 1×TAE buffer by FIGE/U9 electrophoresis. In some cases the REPLI-g mini kit (Qiagen) was used for the amplification following the manufacturer's instructions for purified genomic DNA. Five microliters of yeast plasmid preparation were used as template in an amplification reaction of 50 μl. Reactions were incubated overnight at 30° C. Five to ten microliters of amplified DNA were digested with the restriction enzyme NotI in 50 μl volume at 37° C. for one hour. Twenty microliters of the digest were separated on a 1% agarose gel with EtBr in 1×TAE buffer by FIGE/U9 electrophoresis (FIGS. 18A-18B).

FIGS. 18A-18B show rolling circle amplification (RCA) products derived from the HMG eighth molecule assemblies. FIG. 18A shows supercoil DNA extracted from yeast clones containing the HMG eighth molecule assemblies and used as template in RCA reaction with GE-Templiphi Large Construct kit. FIG. 18B shows supercoil DNA extracted from yeast clones containing the HMG eighth molecule assemblies and used as template in RCA reaction with Qiagen-REPLI-g kit. The RCA products were digested with NotI and separated on an agarose gel subjected to field-inverted gel electrophoresis (FIGE) using the U-9 program as previously described in Gibson et al., Science 319:1215-1220 (2008), which is hereby incorporated by reference. The expected insert size for each eighth molecule is indicated above each lane.

FIG. 19 shows field-inverted gel electrophoresis analysis of HMG. Yeast clones harboring HMG (lane 2) was purified from yeast in agarose plugs, digested with AscI to linearize the 483-kb genome, and then analyzed by FIGE using the U-2 program, which has been described in Gibson et al., Science 319:1215-1220 (2008). The same analysis was performed with yeast not harboring HMG as a negative control (lane 1). M indicates the lambda ladder (NEB).

Cassette Manipulations.

In some cases, sequence-verified cassettes from HMG, RGD1, and RGD2 were further manipulated to match the present design. FIG. 20 illustrates how this was performed. Cassettes were PCR amplified upstream and downstream of a site of insertion or deletion. Genes to be added back were amplified using JCVI-Syn1.0 genomic DNA as template. To make a base substitution, the change was made within the PCR primer. Vector and insert DNA fragments were designed such that they contained 40 bp overlaps to facilitate in vitro DNA assembly. Newly assembled cassettes were sequence verified following cloning, as described above, prior to assembly in yeast to generate the new version of the respective ⅛^(th) molecule.

FIG. 20 is a schematic illustration showing the editing of previously generated sequence-verified cassettes. Cassettes (black rectangles) generated during the construction of HMG, RGD1, and RGD2 were manipulated to remove genes (white square), add genes (dark grey square), and make single nucleotide substitutions (light grey circle). This was readily performed by generating overlapping fragments via PCR (black arrows) and then assembling the resulting fragments in vitro.

PacBio Complete Genome De Novo Assemblies.

An alkaline-lysis approach followed by phenol extraction and ethanol precipitation was used to isolate high molecular weight DNA from Syn3.0 transplants. DNA was quantitated (Qubit, Thermo Fisher Scientific) and quality controlled using an E-Gel (Thermo Fisher Scientific) and then purified using AMPure PB (Pacific Biosciences). The samples (approximately 8-10 μg) were then sheared to an average of 8-20 kb using a g-TUBE (Covaris) at 4500 RPM in an Eppendorf 5424 centrifuge. The samples were then cleaned (Power Clean Pro DNA Clean-Up kit, Mo Bio), quantitated (Qubit, Thermo Fisher Scientific) and quality controlled (Bioanalyzer, Agilent). Adhering to the Pacific Biosciences template preparation protocol (SMRTbell Template Prep Kit 1.0), the samples were first treated with Exo VII to remove single-stranded ends from the DNA fragments, and then taken through DNA damage and end repair before being re-purified using AMPure PB beads. SMRTbell adapter ligation was then performed overnight and failed ligation products were removed with Exo III and Exo VII. AMPure purified DNA was again quantitated (Qubit, Thermo Fisher Scientific) and quality controlled (Bioanalyzer, Agilent) before being size selected (2 or 8 kb to 50 kb) using the BluePippin (0.75%, DF Marker 51 high pass 6-10 kb v3, Sage Science). The size selection was then AMPure PB purified and verified on the Bioanalyzer (Agilent). Sequencing primer was then annealed to the size-selected SMRTbell templates followed by polymerase binding (DNA/Polymerase Binding Kit P6 v2). The prepared libraries were then bound to magnetic beads and loaded onto the Pac Bio RS II at a concentration of 0.200 nM (DNA Sequencing Kit 4.0, Pacific Biosciences). Between 210 and 760 MB of data was generated using one SMRT Cell (V3) per library. Reads of insert ranged from 3700 bp and 9500 bp with polymerase lengths ranging from 11,000 bp to 15,000 bp.

Each sample was assembled de novo using SMRT Analysis 2.3.0 RS_HGAP_Assembly.3 protocol. In short, subreads were extracted using the standard SMRT Analysis 2.3.0 P_Filter protocol using readScore=0.75 and minSubReadLength=500 yielding between 709 and 1151 Mbp of filtered subreads per sample with mean subread lengths between 3475 and 8427 bp. Subreads were then error corrected using the P_PreAssemblerDagcon module using computeLengthCutoff=True and genomeSize=600000 yielding between 6.3 and 9.9 Mbp error corrected reads per sample with N50 lengths between 8780 and 24344 bp. Error corrected reads were assembled to unitigs using the P_AssembleUnitig module and polished using the P_AssemblyPolishing module both using default parameters. The assembly resulted in a single circular contig matching the expected Syn3.0 reference size. Overlapping regions on the 5′ and 3′ end of the circular contigs were later manually trimmed using CLC Genomics Workbench, finalizing the complete genome. In order to identify the differences between the expected and assembled genomes, each assembled reference genome was mapped to its corresponding expected reference using BWA-mem Version: 0.7.12-r1039 with default settings. Variants were then called with CLC Genomics Workbench 8.0.2 using the Basic Variant Detection tool with “Minimum coverage=1” and “Minimum count=1”, resulting in 9 to 27 variant calls per sample. These variants were confirmed using Illumina MiSeq 2×250 bp reads (processed as described above).

FIG. 21 illustrates the general approach used for whole genome synthesis and assembly using HMG as an example. Overlapping oligonucleotides were designed, chemically synthesized, and assembled into 1.4-kb fragments (white). Following error correction and PCR amplification, five fragments were assembled into 7-kb cassettes (black). Cassettes were sequence verified and then assembled in yeast to generate eighth molecules (dark grey). The eight molecules were amplified by RCA and then assembled in yeast to generate the complete genome (light grey).

An automated genome synthesis protocol was established to generate overlapping oligonucleotide sequences starting from a DNA sequence design. Briefly, the software parameters included the number of assembly stages, overlap length, maximum oligonucleotide size, and appended sequences to facilitate PCR amplification or cloning and hierarchical DNA assembly. Approximately 48 oligonucleotides were pooled, assembled, and amplified to generate 1.4-kb DNA fragments in a single reaction (FIGS. 16A-16B, 17). The 1.4-kb DNA fragments were then error corrected, re-amplified, assembled five-at-a-time into a vector, and then transformed into E. coli. Error-free 7-kb cassettes were identified on an Illumina MiSeq DNA sequencer and as many as 15 cassettes were assembled in yeast to generate ⅛^(th) molecules. Supercoiled plasmid DNA was prepared from positively-screened yeast clones and rolling circle amplification was performed to generate microgram quantities of DNA for whole-genome assembly, which was performed again in yeast (FIGS. 18A-18B, 19, 20).

Example 1 Knowledge-Based “Hypothetical Minimal Genome” (HMG) Design

This example described a knowledge-based design of “Hypothetical Minimal Genome” (HMG), which was 483 kb in size and contained 432 protein genes and 39 RNA genes.

M. mycoides JCVI-Syn1.0 (1,078,809 bp, referred herein as “Syn1.0”) described in Gibson et al., Science 329:52-6 (2010), the content of which is hereby incorporated by reference in its entirety) was used as a starting point to design and create a minimized cell. The genome of JCVI-Syn1.0 is virtually identical as the wild-type M. mycoides genome, with a few watermark and vector sequences added. The first step of rational minimal cell design was to design a genome of reduced size from Syn1.0 based on available knowledge, including biochemical literature and some transposon mutagenesis data which consisted of approximately 16,000 Tn4001 and Tn5 insertions into the Syn1.0 genome. With this information a total of 440 apparently non-essential genes were found and deleted from the Syn1.0 genome. The resulting “Hypothetical Minimal Genome” design (HMG) was 483 kb in size and contained 432 protein genes and 39 RNA genes (Table 8 shows a detailed gene list).

TABLE 8 List of genes kept in various genome designs. Table 10. The protein genes and 39 RNA genes in HMG, Syn2.0, Syn3.0, RGD1, and RGD2. Begin. The first nucleotide that is also part of the gene. As with genbank nomenclature, this may be the first nucleotide of the start codon or the complement of the last nucleotide of the stop codon. Numbering according to accession CP002027. End. The last nucleotide that is also part of the gene. As with genbank nomenclature, this may be the complement of the first nucleotide of the start codon or the last nucleotide of the stop codon. Numbering according to accession CP002027. Direction. Forward and reverse. Direction according to accession CP002027 Fragment. From 1 to 8 the numbers indicate which HMG and RGD fragment the gene is part of. Locus tag (accession CP002027) MMSYN1_x KeepDelete. HMG design. k = keep, d = delete, j = other, r = RNA KeepDelete. Syn2.0. k = keep, d = delete, j = other, r = RNA KeepDelete. Syn3.0. k = keep, d = delete, j = other, r = RNA KeepDelete. RGD1 design. k = keep, d = delete, j = other, r = RNA KeepDelete. RGD2 design. k = keep, d = delete, j = other, r = RNA Essential? Date = 130821 Essential? Date = 150126 Current annotation Functional classification Functional category 4064 5155 f 1 0005 k k k k k in n hypothetical protein 1 = Unclear Unknown 151368 151808 r 2 0116 k k k k k ie i hypothetical protein 1 = Unclear Unknown 177917 179815 f 2 0138 d k k k k in i hypothetical protein 1 = Unclear Unknown 183353 184825 f 2 0143 k k k k k e e membrane protein, 1 = Unclear putative Unknown 186592 187299 f 2 0146 k k k k k e e hypothetical protein 1 = Unclear Unknown 212293 212787 r 2 0164 k k k k k ie i hypothetical protein 1 = Unclear Unknown 303170 303460 f 3 0235 k k k k k in in hypothetical protein 1 = Unclear Unknown 305488 307275 f 3 0239 d k k k k i i hypothetical protein 1 = Unclear Unknown 323909 324469 r 3 0248 k k k k k ie ie hypothetical protein 1 = Unclear Unknown 324471 325049 r 3 0249 k k k k k e n? hypothetical protein 1 = Unclear Unknown 325052 325741 r 3 0250 d k k k k n i hypothetical protein 1 = Unclear Unknown 360428 361108 f 3 0281 d k k k k i e hypothetical protein 1 = Unclear Unknown 365884 366690 f 3 0286 k k k k k e n hypothetical protein 1 = Unclear Unknown 376464 377108 f 3 0296 k k k k k e e? hypothetical protein 1 = Unclear Unknown 379011 379310 r 3 0298 k k k k k e i hypothetical protein 1 = Unclear Unknown 379297 379578 r 3 0299 k k k k k e i PF04296 family 1 = Unclear protein Unknown 381929 382579 r 3 0302 k k k k k e ie hypothetical protein 1 = Cofactor Unknown transport and salvage 400483 400635 f 4 0315 k k k k k e i hypothetical protein 1 = Unclear Unknown 402680 402925 f 4 0317 k k k k k e e PF03672 family 1 = Unclear protein Unknown 415769 416530 f 4 0326 k k k k k in n hypothetical protein 1 = Unclear Unknown 437411 438106 f 4 0346 d k k k k ie i hypothetical protein 1 = Unclear Unknown 442525 442902 f 4 0353 d k k k k in i hypothetical protein 1 = Unclear Unknown 462590 464197 f 4 0373 d k k k k e e hypothetical protein 1 = Unclear Unknown 464383 465123 f 4 0375 k k k k k e n hypothetical protein 1 = Unclear Unknown 465125 465439 f 4 0376 d k k k k in in hypothetical protein 1 = Unclear Unknown 467567 467845 f 4 0379 k k k k k e e hypothetical protein 1 = Unclear Unknown 474783 475400 f 4 0388 k k k k k e e hypothetical protein 1 = Unclear Unknown 475476 477620 f 4 0389 d k k k k i i PF11074 domain 1 = Unclear protein Unknown 479222 479524 f 4 0392 k k k k k e n hypothetical protein 1 = Unclear Unknown 489280 491742 r 4 0398 d k k k k i ie lipoprotein, putative 1 = Lipoprotein Unknown 509471 510985 f 4 0411 d k k k k i i membrane protein, 1 = Unclear PF02588 family Unknown 521435 521824 r 5 0416 k k k k k in n hypothetical protein 1 = Unclear Unknown 525808 526119 r 5 0421 k k k k k in i alkaline shock 1 = Unclear protein Asp23 Unknown family protein 526716 527159 f 5 044 k k k k k ie n hypothetical protein 1 = Unclear Unknown 535751 536434 f 5 0433 d k k k k i i CutC family protein 1 = Unclear Unknown 540829 542985 r 5 0439 d k k k k i e lipoprotein, putative 1 = Lipoprotein Unknown 542985 545921 r 5 0440 d k k k k i i lipoprotein, putative 1 = Lipoprotein Unknown 585152 585736 r 5 0478 k k k k k e e hypothetical protein 1 = Unclear Unknown 589611 590048 r 5 0481 k k k k k ie n lipoprotein, putative 1 = Lipoprotein Unknown 608468 608731 r 5 0500 k k k k k e e PF04327 family 1 = Unclear protein Unknown 612638 613261 r 5 0511 d k k k k i e hypothetical protein 1 = Unclear Unknown 616368 617669 r 5 0516 d k k k k i i membrane protein, 1 = Unclear putative Unknown 632115 632723 r 6 0530 k k k k k ie ie hypothetical protein 1 = Unclear Unknown 632732 637180 r 6 0531 d k k k k i e efflux 1 = Efflux Unknown 740801 740941 r 6 0599 k k k k k ie n hypothetical protein 1 = Unclear Unknown 774071 774547 r 6 0632 d k k k k ie i hypothetical protein 1 = Unclear Unknown 778965 781754 r 6 0636 d k k k k e e lipoprotein, putative 1 = Lipoprotein Unknown 830816 831571 r 7 0696 k k k k k ie ie RDD family protein 1 = Unclear Unknown 861788 862387 f 7 0730 k k k k k n? in hypothetical protein 1 = Unclear Unknown 914429 914914 f 7 0777 k k k k k i in hypothetical protein 1 = Unclear Unknown 915034 915285 f 7 0778 k k k k k e e hypothetical protein 1 = Unclear Unknown 936077 936313 r 7 0797 d k k k k e e hypothetical protein 1 = Unclear Unknown 973981 975447 f 8 0827 d k k k k i i hypothetical protein 1 = Unclear Unknown 976624 976884 f 8 0830 k k k k k ie ie hypothetical protein 1 = Unclear Unknown 981034 982341 f 8 0835 k k k k k ie e lipoprotein, putative 1 = Lipoprotein Unknown 1004037 1004219 r 8 0851 d k k k k in n lipoprotein, putative 1 = Lipoprotein Unknown 1004385 1004615 r 8 0852 k k k k k in n hypothetical protein 1 = Unclear Unknown 1004605 1005324 r 8 0853 k k k k k i n hypothetical protein 1 = Unclear Unknown 1029540 1029740 r 8 0873 k k k k k ie n? PF06107 family 1 = Unclear protein Unknown 50958 54116 r 1 0033 k k k d k n ie hypothetical protein 1 = Unclear Unknown 85704 86528 f 1 0060 d k k d k n i membrane protein, 1 = Unclear putative Unknown 109294 109515 r 1 0080 d k k d k in n PF09954 family 1 = Unclear protein Unknown 420267 421094 f 4 0332 d k k d k n i hypothetical protein 1 = Unclear Unknown 426379 427098 f 4 0338 d k k d k n i lipoprotein, putative 1 = Lipoprotein Unknown 609233 609634 r 5 0503 k k k d d in in hypothetical protein 1 = Unclear Unknown 44998 45597 f 1 0029 k k k k k e ie FMN-dependent 2 = Unclear NADH-azoreductase 1 Generic 45634 46668 r 1 0030 k k k k k ie e ABC transporter, 2 = Transport ATP-binding protein Generic 54281 59650 f 1 0034 d k k k k i i efflux ABC 2 = Efflux transporter, Generic permease protein 65087 67033 r 1 0039 k k k k k e e ftsH peptidase? 2 = Protein export Generic 69302 70129 r 1 0042 k k k k k in i transcriptional 2 = Regulation regulator, RpiR Generic family 70181 70903 r 1 0043 k k k k k in n ribosomal protein 2 = rRNA L11 Generic modification methyltransferase- like protein 88297 89271 f 1 0063 k k k k k n n putative tRNA- 2 = tRNA dihydrouridine Generic modification synthase B 91185 92027 r 1 0066 d k k k k i i Cof-like hydrolase 2 = Unclear Generic 126974 127690 r 1 0094 d k k k k n in membrane protein, 2 = Unclear putative Generic 131380 132291 f 1 0097 d k k k k i i 5′-3′ exonuclease, N- 2 = DNA metabolism terminal resolvase- Generic like domain protein 142042 143154 r 2 0108 k k k k k i i lipoprotein, putative 2 = Lipoprotein Generic 143180 144049 r 2 0109 k k k k k ie e apurinic 2 = DNA repair endonuclease Generic (APN1)? 165521 166735 f 2 0127 d k k k k in i HD domain protein 2 = Unclear Generic 171185 172246 f 2 0132 d k k k k i i ATPase, AAA 2 = Unclear family Generic 172239 174512 f 2 0133 d k k k k i n peptidase, S8/S53 2 = Proteolysis family Generic 179825 180775 f 2 0139 d k k k k i e DHHA1 domain 2 = Unclear protein Generic 185449 186363 f 2 0145 k k k k k ie ie Acetyltransferase, 2 = Unclear GNAT family Generic 213005 214249 f 2 0165 k k k k k i i AmiC? 2 = Transport Generic 214265 215275 f 2 0166 k k k k k i i AmiD? 2 = Transport Generic 215289 216989 f 2 0167 k k k k k i i AmiE? 2 = Transport Generic 216991 218859 f 2 0168 k k k k k i i AmiF? 2 = Transport Generic 218876 221977 f 2 0169 d k k k k i i AmiA? 2 = Lipoprotein Generic 257213 260323 r 3 0195 k k k k k n i potCD or potHI? 2 = Lipoprotein Generic 260308 261300 r 3 0196 k k k k k i i potB or potG? 2 = Transport Generic 261300 262355 r 3 0197 k k k k k i i potA or potF? 2 = Transport Generic 278321 278752 f 3 0215 k k k k k e ie RNAse H domain 2 = Unclear protein, YqgF Generic family 341011 342126 r 3 0264 d k k k k in in kinase domain 2 = Unclear protein Generic 362526 363143 f 3 0283 k k k k k ie ie caulimovirus 2 = DNA replication viroplasmin/ Generic ribonuclease HI multi-domain protein 388276 389352 r 3 0305 d k k k k in n metallopeptidase 2 = Proteolysis family M24 Generic 399627 400361 f 4 0314 k k k k k i ie ecfS 2 = Cofactor Generic transport and salvage 414261 415751 f 4 0325 k k k k k i ie membrane protein, 2 = Unclear putative Generic 416540 417376 f 4 0327 k k k k k ie ie scpA? 2 = Chromosome Generic segregation 436455 437345 f 4 0345 k k k k k e ie ecfS 2 = Cofactor Generic transport and salvage 441114 441386 f 4 0350 k k k k k e e putative DNA- 2 = Unclear binding protein HU Generic 1 441441 441989 f 4 0352 k k k k k in e? dnaD? 2 = Unclear Generic 458816 460687 f 4 0371 k k k k k i e ABC transporter, 2 = Efflux ATP-binding protein Generic 460703 462556 f 4 0372 d k k k k i e ABC transporter, 2 = Efflux ATP-binding protein Generic 469634 470272 f 4 0382 k k k k k in n deoxynucleoside 2 = Nucleotide kinase Generic salvage 491892 497153 f 4 0399 d k k k k i ie efflux ABC 2 = Efflux transporter, Generic permease protein 497201 497752 f 4 0400 d k k k k in i DJ-1 family protein 2 = Ribosome Generic biogenesis 497850 499634 f 4 0401 k k k k k in in peptidase, C39 2 = Proteolysis family Generic 506621 507298 f 4 0408 k k k k k i n tRNA: m1A22 2 = tRNA methyltransferase? Generic modification 507285 508061 f 4 0409 d k k k k in in folE? 2 = Cofactor Generic transport and salvage 508070 509431 f 4 0410 d k k k k i i DEAD/DEAH box 2 = Ribosome helicase Generic biogenesis 524140 525783 r 5 0420 k k k k k e e DAK2 domain 2 = Unclear fusion protein YloV Generic 533291 533626 f 5 0430 k k k k k e n Sigma3 and sigma4 2 = Regulation domains of RNA Generic polymerase sigma factors? 533614 534387 f 5 0431 k k k k k e ie putative 2 = Unclear metallophosphoesterase Generic 539417 540397 f 5 0437 k k k k k e e putative 3′-5′ 2 = RNA metabolism exoribonuclease Generic YhaM 540428 540826 r 5 0438 k k k k k e? n Histidine triad (HIT) 2 = Unclear hydrolase-like Generic protein 552275 552778 f 5 0447 k k k k k e e dUTP 2 = Nucleotide diphosphatase? Generic salvage 555218 556633 f 5 0451 k k k k k e e putative 2 = Glucose transport glyceraldehyde-3- Generic & catabolism phosphate dehydrogenase (NADP+) 585741 587387 r 5 0479 d k k k k i i peptidase 2 = Proteolysis Generic 601858 603207 r 5 0493 d k k k k i ie putative dipeptidase 2 = Proteolysis Generic 743662 744606 f 6 0601 d k k k k n i membrane protein, 2 = Unclear putative Generic 761690 762298 r 6 0615 k k k k k i i tRNA binding 2 = Unclear domain protein Generic 764373 764837 r 6 0620 k k k k k i i transcription factor, 2 = Regulation Fur family Generic 782896 787041 r 6 0639 k k k k k e e efflux ABC 2 = Efflux transporter, Generic permease protein 787136 787891 r 6 0640 k k k k k n in putative tRNA 2 = tRNA pseudouridine(38- Generic modification 40) synthase 824338 826359 r 7 0691 k k k k k e e membrane protein, 2 = Efflux putative Generic 826362 827270 r 7 0692 k k k k k n n pseudouridine 2 = rRNA synthase, RluA Generic modification family 827273 828241 r 7 0693 k k k k k ie ie CAAX protease 2 = Proteolysis Generic 831571 832527 r 7 0697 d k k k k i n glycosyltransferase, 2 = Unclear group 2 family Generic protein 848530 849393 r 7 0710 d k k k k i i Cof-like hydrolase 2 = Unclear Generic 859254 860090 f 7 0728 k k k k k in i HAD hydrolase, 2 = Unclear family IIB Generic 949315 950490 r 8 0805 d k k k k i i transcription factor 2 = Regulation Generic 960185 961114 r 8 0817 d k k k k i i transcription factor, 2 = Regulation WhiA like Generic 966098 966772 r 8 0822 k k k k k i i ecfS 2 = Cofactor Generic transport and salvage 982483 983409 f 8 0836 k k k k k e e ecfS 2 = Cofactor Generic transport and salvage 984745 985479 f 8 0838 k k k k k in i RNA 2 = rRNA methyltransferase, Generic modification TrmH family, group 3 1025896 1027662 r 8 0870 k k k k k i e C4-dicarboxylate 2 = Transport anaerobic carrier Generic 1028410 1029504 r 8 0872 k k k k k i i GTP-binding protein 2 = Ribosome YchF Generic biogenesis 1031135 1032715 r 8 0876 k k k k k i i amino acid permease 2 = Transport Generic 1032862 1033542 r 8 0877 d k k k k i e membrane protein, 2 = Unclear putative Generic 1033514 1035007 r 8 0878 k k k k k i i amino acid permease 2 = Transport Generic 1035110 1037767 r 8 0879 d k k k k i n putative magnesium- 2 = Transport importing ATPase Generic 1039138 1040565 r 8 0881 d k k k k i e membrane protein, 2 = Unclear putative Generic 1075251 1075967 r 1 0906 d k k k k in i choline/ethanolamine 2 = Lipid salvage and kinase? Generic biogenesis 1075967 1076761 r 1 0907 d k k k k in n Cof-like hydrolase 2 = Unclear Generic 80000 80449 r 1 0054 d k k d k in n redoxin 2 = Redox Generic homeostasis 106239 107078 r 1 0077 k k k d k n n Cof-like hydrolase 2 = Unclear Generic 548379 550274 f 5 0444 d k k d k n n peptidase family 2 = Proteolysis M13 Generic 604102 604977 r 5 0495 k k k d d in n ROK family protein 2 = Regulation Generic 609657 610544 r 5 0504 d k k d k in i 16S rRNA 2 = rRNA (cytidine(1402)- Generic modification 2′-O)- methyltransferase? 610920 611993 f 5 0505 d k k d d n i lipoprotein, putative 2 = Lipoprotein Generic 9991 10968 r 1 0008 d k k k k i e rnsD 3 = Transport Putative 10968 13535 r 1 0009 d k k k k i ie rnsC 3 = Transport Putative 13525 15141 r 1 0010 d k k k k i e rnsA 3 = Transport Putative 15153 16799 r 1 0011 d k k k k i ie rnsB 3 = Lipoprotein Putative 147625 148869 f 2 0113 d k k k k in ie glycosyltransferase, 3 = Lipid salvage and group 2 family Putative biogenesis protein 148943 149863 r 2 0114 d k k k k in i glycosyltransferase, 3 = Lipid salvage and group 2 family Putative biogenesis protein 277498 278319 f 3 0214 d k k k k in i phosphatidylglycero- 3 = Lipid salvage and phosphatase Putative biogenesis 335106 335903 r 3 0259 k k k k k e e nadK 3 = Cofactor Putative transport and salvage 371137 371691 f 3 0291 k k k k k e ie ribF 3 = Cofactor Putative transport and salvage 381322 381816 r 3 0301 k k k k k ie i rimP 3 = Ribosome Putative biogenesis 387049 388077 r 3 0304 k k k k k e e cdsA transferase 3 = Lipid salvage and Putative biogenesis 546124 547362 f 5 0441 k k k k k e e iscS 3 = tRNA Putative modification 547365 547802 f 5 0442 k k k k k e e iscU 3 = tRNA Putative modification 552795 553562 f 5 0448 d k k k k i ie trmH-like 3 = rRNA Putative modification 613310 614248 r 5 0512 k k k k k e e plsC 3 = Lipid salvage and Putative biogenesis 617662 618594 r 5 0517 k k k k k in in rluD 3 = rRNA Putative modification 618575 619183 r 5 0518 k k k k k ie ie lspA 3 = Protein export Putative 625316 626476 r 6 0523 d k k k k i i ftsA 3 = Cell division Putative 751320 752504 r 6 0609 k k k k k e e dnaB 3 = DNA replication Putative 762373 763212 f 6 0616 d k k k k i e fakB 3 = Lipid salvage and Putative biogenesis 763223 764074 f 6 0617 d k k k k i i fakB 3 = Lipid salvage and Putative biogenesis 816697 818298 r 7 0685 k k k k k i e ktrAB 3 = Transport Putative 818377 819105 f 7 0686 d k k k k i e trkA 3 = Transport Putative 844369 846117 r 7 0706 k k k k k i i ABC transporter, 3 = Cofactor permease protein Putative transport and salvage 846081 846830 r 7 0707 k k k k k e i ABC transporter, 3 = Transport ATP-binding protein Putative 846844 848307 r 7 0708 k k k k k i i high affinity 3 = Lipoprotein transport system Putative protein p37 863454 864122 r 7 0732 d k k k k in n deoC: deoxyribose- 3 = Metabolic phosphate aldolase Putative process 937338 938579 r 8 0799 d k k k k i i glyA, transferase 3 = Cofactor Putative transport and salvage 961127 962557 r 8 0818 k k k k k ie ie lgt 3 = Lipid salvage and Putative biogenesis 963482 965062 r 8 0820 k k k k k e e lgt 3 = Lipid salvage and Putative biogenesis 1045710 1047287 r 8 0886 d k k k k i i gltP 3 = Transport Putative 1047307 1048650 r 8 0887 d k k k k i i pyridine nucleotide- 3 = Redox disulfide Putative homeostasis oxidoreductase 197743 199098 f 2 0154 d k k d d in i cytosol 3 = Proteolysis aminopeptidase Putative family, catalytic domain protein 603363 604043 r 5 0494 k k k d k in n putative N- 3 = Transport acetylmannosamine- Putative 6-P epimerase 43531 43971 r 1 0026 k k k k k e e priB 4 = DNA replication Probable 70903 71643 r 1 0044 k k k k k e e DNA polymerase III 4 = DNA replication delta subunit Probable 140016 140231 r 2 0105 k k k k k e i xseB 4 = DNA metabolism Probable 150311 151183 r 2 0115 k k k k k i i galU 4 = Lipid salvage and Probable biogenesis 166761 167189 f 2 0128 d k k k k i i RNA polymerase 4 = Transcription delta subunit Probable 180777 181406 f 2 0140 k k k k k i ie tdk 4 = Nucleotide Probable salvage 184834 185334 f 2 0144 k k k k k e i tsaC 4 = tRNA Probable modification 292935 294260 f 3 0227 d k k k k i i pdhC 4 = Metabolic Probable process 300902 302623 f 3 0233 k k k k k ie ie phosphoenolpyruvate- 4 = Glucose transport protein Probable & catabolism phosphotransferase 302705 303169 f 3 0234 k k k k k ie e glucose-specific 4 = Glucose transport phosphotransferase Probable & catabolism enzyme IIA component 307283 308470 f 3 0240 k k k k k i i thiI 4 = tRNA Probable modification 327283 327756 r 3 0253 k k k k k ie e greA 4 = Transcription Probable 332498 334249 r 3 0257 k k k k k ie ie rnjB 4 = RNA metabolism Probable 418057 418815 f 4 0329 k k k k k i n Ribosomal large 4 = rRNA subunit Probable modification pseudouridine synthase B 418825 419442 f 4 0330 k k k k k i n dgk 4 = Nucleotide Probable salvage 435756 436316 r 4 0344 k k k k k e e Ppase 4 = Metabolic Probable process 505101 506621 f 4 0407 k k k k k e e rpoD 4 = Transcription Probable 511101 515255 f 4 0412 k k k k k e e secDF 4 = Protein export Probable 515862 518126 f 4 0414 d k k k k i n relA 4 = Regulation Probable 527248 528378 f 5 0425 k k k k k i e pstS 4 = Lipoprotein Probable 550494 551777 f 5 0445 k k k k k e e gpi 4 = Glucose transport Probable & catabolism 614414 614746 r 5 0513 k k k k k e e acpS 4 = Lipid salvage and Probable biogenesis 615883 616365 r 5 0515 k k k k k i n dctD 4 = Nucleotide Probable salvage 651017 651619 r 6 0543 k k k k k e e grpE 4 = Protein folding Probable 652648 654789 r 6 0545 d k k k k ie ie clpB 4 = Proteolysis Probable 741755 743587 f 6 0600 k k k k k e e rnjA 4 = RNA metabolism Probable 747929 749143 r 6 0606 k k k k k e e pgk 4 = Glucose transport Probable & catabolism 750372 751310 r 6 0608 k k k k k e e dnaI 4 = DNA replication Probable 756240 759203 r 6 0612 k k k k k e ie dnaE 4 = DNA replication Probable 760651 761712 r 6 0614 k k k k k e e pncB 4 = Cofactor Probable transport and salvage 764838 765059 r 6 0621 k k k k k ie e acpA 4 = Lipid salvage and Probable biogenesis 781913 782311 r 6 0637 k k k k k e e rpsE Probable Translation 787894 788904 r 6 0641 k k k k k e e ecfT 4 = Cofactor Probable transport and salvage 788918 789829 r 6 0642 k k k k k e e ecfA 4 = Cofactor Probable transport and salvage 789817 791043 r 6 0643 k k k k k e e ecfA 4 = Cofactor Probable transport and salvage 794477 795118 r 6 0651 k k k k k e e adk 4 = Nucleotide Probable salvage 798865 799254 r 6 0657 k k k k k e e rpsH 4 = Translation Probable 799274 799459 r 6 0658 k k k k k e e rpsN 4 = Translation Probable 799478 800020 r 6 0659 k k k k k e e rplE 4 = Translation Probable 804121 804405 r 6 0669 k k k k k e e rplW 4 = Translation Probable 815822 816688 r 7 0684 d k k k k i e folD 4 = Cofactor Probable transport and salvage 820573 822030 r 7 0688 k k k k k e e gatA 4 = Translation Probable 828297 828566 r 7 0694 k k k k k e ie ptsH 4 = Glucose transport Probable & catabolism 828631 830799 r 7 0695 d k k k k i i pcrA 4 = DNA replication Probable 864134 865810 r 7 0733 d k k k k i i pgcA 4 = Lipid salvage and Probable biogenesis 878368 879021 f 7 0747 d k k k k i i punA 4 = Nucleotide Probable salvage 910221 911240 r 7 0773 d k k k k i ie nrdF 4 = Nucleotide Probable salvage 915336 917573 f 7 0779 d k k k k ie ie ptsG 4 = Glucose transport Probable & catabolism 929590 929889 r 7 0789 k k k k k ie ie atpC 4 = Transport Probable 929889 931316 r 7 0790 k k k k k i i atpD 4 = Transport Probable 935214 936077 r 7 0796 k k k k k ie e atpB 4 = Transport Probable 951098 951595 r 8 0807 k k k k k e e rplJ 4 = Translation Probable 966789 967904 r 8 0823 k k k k k i n folC 4 = Cofactor Probable transport and salvage 973028 973978 f 8 0826 d k k k k e e yqeN 4 = DNA replication Probable 977064 978098 f 8 0831 k k k k k e i prs 4 = Nucleotide Probable salvage 986041 986682 f 8 0840 k k k k k i e nusG 4 = Transcription Probable 1012008 1013969 f 8 0859 k k k k k e i topA 4 = DNA topology Probable 1076838 1078028 r 1 0908 k k k k k e e misC 4 = Protein export Probable 75393 75491 r 1 0049 r r r r r e e srpB 5 = RNA Equivalog 92148 92256 r 1 0067 r r r r r ie i 5S rRNA 5 = RNA Equivalog 92331 95225 r 1 0068 r r r r r ie i 23S rrna 5 = RNA Equivalog 95457 96980 r 1 0069 r r r r r ie i 16S rRNA 5 = RNA Equivalog 97263 97346 r 1 0070 r r r r r e e tRNA-Leu 5 = RNA Equivalog 97349 97424 r 1 0071 r r r r r e e tRNA-Lys 5 = RNA Equivalog 203879 204289 f 2 0158 r r r r r e e ssrA 5 = RNA Equivalog 360219 360308 f 3 0280 r r r r r e e tRNA-Ser 5 = RNA Equivalog 376293 376366 f 3 0295 r r r r r e e tRNA-Gly 5 = RNA Equivalog 445958 446302 f 4 0356 r r r r r e e RNAse P 5 = RNA Equivalog 464253 464329 f 4 0374 r r r r r e e tRNA-Arg 5 = RNA Equivalog 526549 526633 f 5 0423 r r r r r e i tRNA-Leu 5 = RNA Equivalog 612086 612174 r 5 0506 r r r r r e e tRNA-Leu 5 = RNA Equivalog 612185 612260 r 5 0507 r r r r r e e tRNA-Lys 5 = RNA Equivalog 612265 612339 r 5 0508 r r r r r e e tRNA-Gln 5 = RNA Equivalog 612346 612429 r 5 0509 r r r r r e e tRNA-Tyr 5 = RNA Equivalog 612436 612511 r 5 0510 r r r r r e e tRNA-Thr 5 = RNA Equivalog 764110 764184 r 6 0618 r r r r r ie ie tRNA-Trp 5 = RNA Equivalog 764222 764297 r 6 0619 r r r r r e e tRNA-Trp 5 = RNA Equivalog 766603 766678 r 6 0624 r r r r r e ie tRNA-His 5 = RNA Equivalog 778816 778892 r 6 0635 r r r r r e e tRNA-Ile 5 = RNA Equivalog 813370 813445 r 7 0678 r r r r r e e tRNA-Thr 5 = RNA Equivalog 813458 813533 r 7 0679 r r r r r e e tRNA-Val 5 = RNA Equivalog 813541 813616 r 7 0680 r r r r r e e tRNA-Glu 5 = RNA Equivalog 813624 813699 r 7 0681 r r r r r e e tRNA-Asn 5 = RNA Equivalog 856724 856800 f 7 0717 r r r r r e e tRNA-Arg 5 = RNA Equivalog 856848 856924 f 7 0718 r r r r r e e tRNA-Pro 5 = RNA Equivalog 856935 857010 f 7 0719 r r r r r e e tRNA-Ala 5 = RNA Equivalog 857015 857091 f 7 0720 r r r r r e e tRNA-Met 5 = RNA Equivalog 857103 857179 f 7 0721 r r r r r e e tRNA-Met 5 = RNA Equivalog 857222 857314 f 7 0722 r r r r r e e tRNA-Ser 5 = RNA Equivalog 857337 857412 f 7 0723 r r r r r e e tRNA-Met 5 = RNA Equivalog 857415 857491 f 7 0724 r r r r r e e tRNA-Asp 5 = RNA Equivalog 857500 857575 f 7 0725 r r r r r e i tRNA-Phe 5 = RNA Equivalog 975613 975687 f 8 0828 r r r r r e e tRNA-Cys 5 = RNA Equivalog 1 1353 f 1 0001 k k k k k e e dnaA 5 = DNA replication Equivalog 1511 2638 f 1 0002 k k k k k e e DNA polymerase 5 = DNA replication III, beta subunit Equivalog 2675 3217 f 1 0003 k k k k k i i rnmV 5 = Ribosome Equivalog biogenesis 3207 4007 f 1 0004 k k k k k in n ksgA 5 = rRNA Equivalog modification 5515 7419 f 1 0006 k k k k k e e gyrB 5 = DNA topology Equivalog 7435 9939 f 1 0007 k k k k k e e gyrA 5 = DNA topology Equivalog 16986 18515 r 1 0012 k k k k k e ie metRS 5 = Translation Equivalog 43284 43511 r 1 0025 k k k k k e e rpsR 5 = Translation Equivalog 43983 44396 r 1 0027 k k k k k e e rpsF 5 = Translation Equivalog 68094 69299 r 1 0040 k k k k k e e tilS 5 = tRNA Equivalog modification 71621 72262 r 1 0045 k k k k k e e tmk 5 = Nucleotide Equivalog salvage 72265 72855 r 1 0046 k k k k k in n recR: recombination 5 = DNA repair protein RecR Equivalog 72857 74863 r 1 0047 k k k k k e e DNA polymerase 5 = DNA replication III, subunit gamma Equivalog and tau 86611 87879 f 1 0061 k k k k k e e serRS 5 = Translation Equivalog 89274 90776 f 1 0064 k k k k k e ie lysRS 5 = Translation Equivalog 90866 91174 r 1 0065 k k k k k e e trxA 5 = Redox Equivalog homeostasis 104866 106230 r 1 0076 k k k k k e ie asnRS 5 = Translation Equivalog 108243 109199 r 1 0079 k k k k k ie ie tsaD tRNA 5 = tRNA threonylcarbamoyl- Equivalog modification adenosine. Found in tRNAs decoding ANN (ile, Met, Thr, Lys, Asn, Ser and Arg). 109715 111073 f 1 0081 k k k k k i i mnmE_trmE_thdF: 5 = tRNA tRNA modification Equivalog modification GTPase TrmE 111124 111369 r 1 0082 k k k k k in i S20: ribosomal 5 = Translation protein S20 Equivalog 127805 130639 r 1 0095 k k k k k in ie secA: preprotein 5 = Protein export translocase, SecA Equivalog subunit 140221 141630 r 2 0106 k k k k k i n xseA, 5 = DNA metabolism exodeoxyribonuclease Equivalog VII, large subunit 141632 142030 r 2 0107 k k k k k i e nusB: transcription 5 = Transcription antitermination Equivalog factor NusB 151878 152639 f 2 0117 k k k k k ie ie plsY 5 = Lipid salvage and Equivalog biogenesis 164067 165518 f 2 0126 k k k k k e ie gluRS 5 = Translation Equivalog 167281 168879 f 2 0129 k k k k k e ie PyrG: CTP synthase 5 = Nucleotide Equivalog salvage 170143 171036 f 2 0131 k k k k k e e fruc_bis_ald_: 5 = Glucose transport fructose-1,6- Equivalog & catabolism bisphosphate aldolase, class II 177566 177844 f 2 0137 k k k k k ie i L31: ribosomal 5 = Translation protein L31 Equivalog 181409 182503 f 2 0141 k k k k k e ie prfA: peptide chain 5 = Translation release factor 1 Equivalog 182496 183344 f 2 0142 k k k k k i i PrmC 5 = Translation Equivalog 187299 188828 f 2 0147 k k k k k e e bac_cardiolipin: 5 = Lipid salvage and cardiolipin synthase Equivalog biogenesis 188952 189371 f 2 0148 k k k k k e e rpsL_bact: 5 = Translation ribosomal protein Equivalog S12 189458 189925 f 2 0149 k k k k k e e rpsG_bact: 5 = Translation ribosomal protein S7 Equivalog 189950 192019 f 2 0150 k k k k k e e EF-G: translation 5 = Translation elongation factor G Equivalog 192151 193338 f 2 0151 k k k k k e e EF-Tu: translation 5 = Translation elongation factor Tu Equivalog 209529 212219 r 2 0163 k k k k k e e alaRS 5 = Translation Equivalog 262729 263088 r 3 0198 k k k k k e e rplT_bact: ribosomal 5 = Translation protein L20 Equivalog 263107 263298 r 3 0199 k k k k k e e L35 5 = Translation Equivalog 263324 263869 r 3 0200 k k k k k e e infC: translation 5 = Translation initiation factor IF-3 Equivalog 264058 264660 r 3 0201 k k k k k e e pept_deformyl: 5 = Translation peptide deformylase Equivalog 264734 265291 f 3 0202 k k k k k i i 16S rRNA 5 = rRNA (guanine(966)- Equivalog modification N(2))- methyltransferase RsmD 265294 266187 f 3 0203 k k k k k e e guanyl_kin: 5 = Nucleotide guanylate kinase Equivalog salvage 276076 277431 f 3 0213 k k k k k e e eno: 5 = Glucose transport phosphopyruvate Equivalog & catabolism hydratase 278758 279330 f 3 0216 d k k k k i i HGPRTase: hypoxanthine 5 = Nucleotide phosphoribosyltransferase Equivalog salvage 283236 284216 f 3 0220 k k k k k e e PFKA_ATP: 6- 5 = Glucose transport phosphofructokinase Equivalog & catabolism 284287 285723 f 3 0221 k k k k k e ie pyruv_kin: pyruvate 5 = Glucose transport kinase Equivalog & catabolism 285988 287907 f 3 0222 k k k k k e e thrRS 5 = Translation Equivalog 294279 296168 f 3 0228 d k k k k i i lipoamide_DH: 5 = Metabolic dihydrolipoyl Equivalog process dehydrogenase 296190 297158 f 3 0229 k k k k k in e pta: phosphate 5 = Metabolic acetyltransferase Equivalog process 297171 298352 f 3 0230 k k k k k in i ackA: acetate kinase 5 = Metabolic Equivalog process 304607 305233 r 3 0238 k k k k k e e rpsD_bact: 5 = Translation ribosomal protein S4 Equivalog 323325 323915 r 3 0247 k k k k k e e GTPase_YsxC: 5 = Ribosome ribosome biogenesis Equivalog biogenesis GTP-binding protein YsxC, bsub homolog is essential 327879 329633 r 3 0254 d k k k k n n uvrC: excinuclease 5 = DNA repair ABC subunit C Equivalog 336014 338632 r 3 0260 k k k k k e e valRS 5 = Translation Equivalog 339335 340097 r 3 0262 d k k k k i i ribulose-phosphate 5 = Metabolic 3-epimerase Equivalog process 340099 341001 r 3 0263 d k k k k i i ribosome small 5 = Ribosome subunit-dependent Equivalog biogenesis GTPase A, bsub rsgA is not essential 344441 344857 f 3 0270 k k k k k e e T6A_YjeE: tRNA 5 = tRNA threonylcarbamoyl Equivalog modification adenosine modification protein YjeE 361089 362513 f 3 0282 k k k k k e e proRS 5 = Translation Equivalog 364056 365858 f 3 0285 k k k k k n i lepA: elongation 5 = Translation factor 4 Equivalog 366707 368431 r 3 0287 k k k k k e ie aspRS 5 = Translation Equivalog 368440 369684 r 3 0288 k k k k k e ie hisRS 5 = Translation Equivalog 369869 370222 f 3 0289 k k k k k ie ie rbfA: ribosome- 5 = Ribosome binding factor A Equivalog biogenesis 370272 371150 f 3 0290 k k k k k n n TruB: tRNA 5 = tRNA pseudouridine(55) Equivalog modification synthase 375967 376233 f 3 0294 k k k k k e i S15_bact: ribosomal 5 = Translation protein S15 Equivalog 377127 378989 r 3 0297 k k k k k e i IF-2: translation 5 = Translation initiation factor IF-2 Equivalog 379559 381313 r 3 0300 k k k k k ie ie nusA 5 = Transcription Equivalog 382592 387040 r 3 0303 k k k k k e e polC_Gram_pos: 5 = DNA replication DNA polymerase Equivalog III, alpha subunit, Gram-positive type 391003 392013 f 4 0308 k k k k k e ie trpRS 5 = Translation Equivalog 400694 402664 f 4 0316 d k k k k i i tkt 5 = Metabolic Equivalog process 417360 417989 f 4 0328 k k k k k e ie segregation and 5 = Chromosome condensation protein Equivalog segregation B 438115 438777 f 4 0347 k k k k k i i cmk: cytidylate 5 = Nucleotide kinase Equivalog salvage 438784 440091 f 4 0348 k k k k k e e GTPase_EngA: 5 = Ribosome ribosome-associated Equivalog biogenesis GTPase EngA 448025 449554 f 4 0359 k k k k k e ie RNase_Y: 5 = RNA metabolism ribonuclease Y Equivalog 449588 450931 f 4 0360 k k k k k e ie ffh: signal 5 = Protein export recognition particle Equivalog protein 450935 451402 f 4 0361 k k k k k e n rlmH 5 = rRNA Equivalog modification 451493 451771 f 4 0362 k k k k k e e ribosomal protein 5 = Translation S16 Equivalog 451803 452297 f 4 0363 k k k k k e e 16S_RimM: 16S 5 = Ribosome rRNA processing Equivalog biogenesis protein RimM 452299 453021 f 4 0364 k k k k k i e trmD: tRNA 5 = tRNA (guanine(37)-N(1))- Equivalog modification methyltransferase 453023 453406 f 4 0365 k k k k k e ie rplS_bact: ribosomal 5 = Translation protein L19 Equivalog 453505 454455 f 4 0366 k k k k k e e GTPase_YlqF: 5 = Ribosome ribosome biogenesis Equivalog biogenesis GTP-binding protein YlqF 465510 466811 f 4 0377 k k k k k e e Obg_CgtA: Obg 5 = Ribosome family GTPase CgtA Equivalog biogenesis 466813 467550 f 4 0378 k k k k k e e NAD+ synthetase 5 = Cofactor Equivalog transport and salvage 467863 468960 f 4 0380 k k k k k e ie nicotinate 5 = Cofactor (nicotinamide) Equivalog transport and nucleotide salvage adenylyltransferase 468965 469624 f 4 0381 k k k k k in n MTA/SAH-Nsdase: 5 = Cofactor MTA/SAH Equivalog transport and nucleosidase salvage 473609 474736 f 4 0387 k k k k k e e trmU: tRNA (5- 5 = tRNA methylaminomethyl- Equivalog modification 2-thiouridylate)- methyltransferase 477610 478563 f 4 0390 k k k k k i ie fmt: methionyl- 5 = Translation tRNA Equivalog formyltransferase 478650 479204 f 4 0391 k k k k k i ie efp: translation 5 = Translation elongation factor P Equivalog 480950 483310 f 4 0394 d k k k k i i lon: endopeptidase 5 = Proteolysis La Equivalog 499639 500133 f 4 0402 k k k k k e ie rRNA maturation 5 = Ribosome RNase YbeY Equivalog biogenesis 500137 501042 f 4 0403 k k k k k i ie era: GTP-binding 5 = Ribosome protein Era Equivalog biogenesis 501042 501791 f 4 0404 k k k k k i n reco: DNA repair 5 = DNA repair protein RecO Equivalog 501855 503225 f 4 0405 k k k k k e e glyRS 5 = Translation Equivalog 503260 505098 f 4 0406 k k k k k e e dnaG 5 = DNA replication Equivalog 515267 515779 f 4 0413 k k k k k e e apt: adenine 5 = Nucleotide phosphoribosyltransferase Equivalog salvage 518163 521129 r 4 0415 k k k k k ie i chromosome 5 = Chromosome segregation protein Equivalog segregation SMC 522415 523113 r 5 0418 k k k k k i e RNaseIII: 5 = RNA metabolism ribonuclease III Equivalog 523103 524107 r 5 0419 k k k k k e e plsX: fatty 5 = Lipid salvage and acid/phospholipid Equivalog biogenesis synthesis protein PlsX 526288 526485 f 5 0422 k k k k k e? n L28: ribosomal 5 = Translation protein L28 Equivalog 528430 530520 f 5 0426 k k k k k i e phosphate ABC 5 = Transport transporter, Equivalog permease protein PstA, phosphate_pstC: phosphate ABC transporter, permease protein PstC 530513 531322 f 5 0427 k k k k k i e phosphate ABC 5 = Transport transporter, ATP- Equivalog binding protein 531331 532005 f 5 0428 k k k k k i e phoU_full: 5 = Transport phosphate transport Equivalog system regulatory protein PhoU 532072 533301 f 5 0429 k k k k k e e ftsY: signal 5 = Protein export recognition particle- Equivalog docking protein FtsY 534597 535760 f 5 0432 d k k k k e i metK: methionine 5 = Cofactor adenosyltransferase Equivalog transport and salvage 536427 537743 f 5 0434 k k k k k i n gid_trmFO: 5 = tRNA tRNA: m(5)U-54 Equivalog modification methyltransferase 537769 538698 f 5 0435 k k k k k i n manA: mannose-6- 5 = Carbon source phosphate Equivalog transport & isomerase, class I catabolism 547795 548358 f 5 0443 k k k k k e? n 5- 5 = Cofactor formyltetrahydrofolate Equivalog transport and cyclo-ligase salvage 556820 558751 f 5 0452 k k k k k e e DNA topoisomerase 5 = DNA topology IV, B subunit Equivalog 558753 561449 f 5 0453 k k k k k e e DNA topoisomerase 5 = DNA topology IV, A subunit Equivalog 582072 583028 r 5 0475 d k k k k i ie L-LDH-NAD: L-actate 5 = Metabolic dehydrogenase Equivalog process 590364 590528 f 5 0482 k k k k k e e S21p: ribosomal 5 = Translation protein S21 Equivalog 608185 608466 r 5 0499 k k k k k e e L27: ribosomal 5 = Translation protein L27 Equivalog 608785 609087 r 5 0501 k k k k k e e L21: ribosomal 5 = Translation protein L21 Equivalog 619174 621921 r 5 0519 k k k k k e e ileRS 5 = Translation Equivalog 626485 627411 r 6 0524 k k k k k i i rsmH, 16S rRNA 5 = rRNA m4C1402 Equivalog modification 627420 627821 r 6 0525 d k k k k i i mraZ 5 = Regulation Equivalog 627928 628107 r 6 0526 k k k k k i i rpmF_bact: 5 = Translation ribosomal protein Equivalog L32 628640 631024 r 6 0528 k k k k k e e pheRS 5 = Translation Equivalog 631033 632085 r 6 0529 k k k k k e e pheRS 5 = Translation Equivalog 642339 644003 r 6 0535 k k k k k e e argRS 5 = Translation Equivalog 644005 644553 r 6 0536 k k k k k e e frr: ribosome 5 = Translation recycling factor Equivalog 644564 645277 r 6 0537 k k k k k e e pyrH_bact: UMP 5 = Nucleotide kinase Equivalog salvage 646016 646903 r 6 0539 k k k k k e e tsf: translation 5 = Translation elongation factor Ts Equivalog 646915 647793 r 6 0540 k k k k k e e rpsB_bact: 5 = Translation ribosomal protein S2 Equivalog 648001 649119 r 6 0541 d k k k k e e DnaJ_bact: 5 = Protein folding chaperone protein Equivalog DnaJ 649183 650958 r 6 0542 k k k k k e e prok_dnaK: 5 = Protein folding chaperone protein Equivalog DnaK 651621 652643 r 6 0544 k k k k k e e hrcA: heat-inducible 5 = Regulation transcription Equivalog represser HrcA 749255 750271 r 6 0607 k k k k k e e GAPDH-I: 5 = Glucose transport glyceraldehyde-3- Equivalog & catabolism phosphate dehydrogenase, type I 753496 756231 r 6 0611 k k k k k e e polA: DNA 5 = DNA replication polymerase I Equivalog 759398 760642 r 6 0613 k k k k k e e tyrRS 5 = Translation Equivalog 776264 778678 r 6 0634 k k k k k e e leuRS 5 = Translation Equivalog 782311 782766 r 6 0638 k k k k k e e ribosomal protein 5 = Translation L13 Equivalog 791151 791510 r 6 0644 k k k k k e e L17: ribosomal 5 = Translation protein L17 Equivalog 791530 792483 r 6 0645 k k k k k e e rpoA: DNA-directed 5 = Transcription RNA polymerase, Equivalog alpha subunit 792487 792876 r 6 0646 k k k k k e e bact_S11: 30S 5 = Translation ribosomal protein Equivalog S11 792902 793267 r 6 0647 k k k k k e e bact_S13: 30S 5 = Translation ribosomal protein Equivalog S13 793304 793417 r 6 0648 k k k k k e e rpmJ_bact: 5 = Translation ribosomal protein Equivalog L36 793486 793710 r 6 0649 k k k k k e e infA: translation 5 = Translation initiation factor IF-1 Equivalog 793722 794477 r 6 0650 k k k k k e e met_pdase_I: 5 = Translation methionine Equivalog aminopeptidase, type I 795246 796694 r 6 0652 k k k k k e e preprotein 5 = Protein export translocase, SecY Equivalog subunit 796694 797131 r 6 0653 k k k k k i e rplO_bact: 5 = Translation ribosomal protein Equivalog L15 797150 797914 r 6 0654 k k k k k e e rpsE_bact: 5 = Translation ribosomal protein S5 Equivalog 797933 798283 r 6 0655 k k k k k e e L18_bact: ribosomal 5 = Translation protein L18 Equivalog 798309 798851 r 6 0656 k k k k k e e L6_bact: ribosomal 5 = Translation protein L6 Equivalog 800039 800365 r 6 0660 k k k k k e e rplX_bact: 5 = Translation ribosomal protein Equivalog L24 800379 800747 r 6 0661 k k k k k e e rplN_bact: 5 = Translation ribosomal protein Equivalog L14 800763 801020 r 6 0662 k k k k k e e S17_bact: 30S 5 = Translation ribosomal protein Equivalog S17 801020 801436 r 6 0663 k k k k k e e L29: ribosomal 5 = Translation protein L29 Equivalog 801436 801849 r 6 0664 k k k k k e e rplP_bact: ribosomal 5 = Translation protein L16 Equivalog 801852 802553 r 6 0665 k k k k k e e rpsC_bact: 5 = Translation ribosomal protein S3 Equivalog 802571 802906 r 6 0666 k k k k k e e rplV_bact: 5 = Translation ribosomal protein Equivalog L22 802930 803196 r 6 0667 k k k k k e e rpsS_bact: 5 = Translation ribosomal protein Equivalog S19 803218 804066 r 6 0668 k k k k k e e rplB_bact: 5 = Translation ribosomal protein L2 Equivalog 804405 805031 r 6 0670 k k k k k e e rplD_bact: 50S 5 = Translation ribosomal protein L4 Equivalog 805044 805715 r 6 0671 k k k k k e e L3_bact: 50S 5 = Translation ribosomal protein L3 Equivalog 805791 806099 r 6 0672 k k k k k e e rpsJ_bact: ribosomal 5 = Translation protein S10 Equivalog 819132 820571 r 7 0687 k k k k k e e gatB: 5 = Translation aspartyl/glutamyl- Equivalog tRNA(Asn/Gln) amidotransferase, B subunit 822030 822326 r 7 0689 k k k k k e e gatC: 5 = Translation aspartyl/glutamyl- Equivalog tRNA(Asn/Gln) amidotransferase, C subunit 822328 824334 r 7 0690 k k k k k e e dnlj: DNA ligase, 5 = DNA replication NAD-dependent Equivalog 857683 858417 f 7 0726 k k k k k ie in nagB: glucosamine- 5 = Carbon source 6-phosphate Equivalog transport & deaminase catabolism 858515 859261 f 7 0727 k k k k k e e tpiA, tim: triose- 5 = Glucose transport phosphate isomerase Equivalog & catabolism 860093 861688 f 7 0729 k k k k k e e pgm_bpd_ind: 5 = Glucose transport phosphoglycerate Equivalog & catabolism mutase (2,3- diphosphoglycerate- independent) 907590 909752 r 7 0771 d k k k k i i NrdE_NrdA: 5 = Nucleotide ribonucleoside- Equivalog salvage diphosphate reductase, alpha subunit 909739 910212 r 7 0772 k k k k k i e NrdI 5 = Nucleotide Equivalog salvage 911539 911823 f 7 0774 k k k k k e e secG: preprotein 5 = Protein export translocase, SecG Equivalog subunit 911859 913973 f 7 0775 d k k k k i i RNase_R: 5 = RNA metabolism ribonuclease R Equivalog 913983 914429 f 7 0776 k k k k k e e smpB: SsrA-binding 5 = Translation protein Equivalog 925156 927984 r 7 0787 k k k k k ie e ATPase-IIIB_Mg: 5 = Transport magnesium- Equivalog translocating P-type ATPase 931325 932167 r 7 0791 k k k k k e e ATPsyn_F1gamma: 5 = Transport ATP synthase F1, Equivalog gamma subunit 932169 933746 r 7 0792 k k k k k i ie atpA: ATP synthase 5 = Transport F1, alpha subunit Equivalog 933758 934303 r 7 0793 k k k k k ie e ATP_synt_delta: 5 = Transport ATP synthase F1, Equivalog delta subunit 934305 934850 r 7 0794 k k k k k ie ie ATP_synt_b: ATP 5 = Transport synthase F0, B Equivalog subunit 934879 935184 r 7 0795 k k k k k ie e ATP_synt_c: ATP 5 = Transport synthase F0, C Equivalog subunit 936615 937238 r 8 0798 d k k k k in i upp: uracil 5 = Nucleotide phosphoribosyltransferase Equivalog salvage 938563 939006 r 8 0800 d k k k k i i rpiB: ribose 5- 5 = Metabolic phosphate isomerase Equivalog process B 941671 945438 r 8 0803 k k k k k e e rpoC_TIGR: DNA- 5 = Transcription directed RNA Equivalog polymerase, beta′ subunit 945450 949325 r 8 0804 k k k k k e e rpoB: DNA-directed 5 = Transcription RNA polymerase, Equivalog beta subunit 950661 951029 r 8 0806 k k k k k e e L12: ribosomal 5 = Translation protein L7/L12 Equivalog 951822 952502 r 8 0809 k k k k k i i rplA_bact: 5 = Translation ribosomal protein L1 Equivalog 952502 952930 r 8 0810 k k k k k i e L11_bact: ribosomal 5 = Translation protein L11 Equivalog 956226 957224 f 8 0813 d k k k k i i galE: UDP-glucose 5 = Lipid salvage and 4-epimerase GalE Equivalog biogenesis 957238 958425 f 8 0814 d k k k k i i glf: UDP- 5 = Lipid salvage and GALP_mutase: Equivalog biogenesis UDP-galactopyranose mutase 962566 963498 r 8 0819 k k k k k e e TRX_reduct: 5 = Redox thioredoxin-disulfide Equivalog homeostasis reductase 965073 966014 r 8 0821 d k k k k in i hpr-ser: HPr(Ser) 5 = Glucose transport kinase/phosphatase Equivalog & catabolism 967897 970737 r 8 0824 d k k k k in n uvra: excinuclease 5 = DNA repair ABC subunit A Equivalog 970746 972743 r 8 0825 d k k k k in n uvrb: excinuclease 5 = DNA repair ABC subunit B Equivalog 978524 979084 f 8 0832 k k k k k e ie pth: aminoacyl- 5 = Translation RNA hydrolase Equivalog 979205 979648 f 8 0833 k k k k k ie n L9: ribosomal 5 = Translation protein L9 Equivalog 979651 980967 f 8 0834 k k k k k e e dnaC 5 = DNA replication Equivalog 983418 984743 f 8 0837 k k k k k e e cysRS 5 = Translation Equivalog 985701 986024 f 8 0839 k k k k k e e secE_bact: 5 = Protein export preprotein Equivalog translocase, SecE subunit 1029742 1030437 r 8 0874 k k k k k n i rsmG_gidB: 5 = rRNA Equivalog modification 1030437 1031033 r 8 0875 k k k k k e e pgsA: CDP- 5 = Lipid salvage and diacylglycerol-- Equivalog biogenesis glycerol-3- phosphate 3- phosphatidyltransferase 1043673 1045562 r 8 0885 k k k k k i i gidA: tRNA uridine 5 = tRNA 5- Equivalog modification carboxymethyl- aminomethyl modification enzyme GidA 1078046 1078375 r 1 0909 k k k k k e e rnpA: ribonuclease P 5 = RNA metabolism protein component Equivalog 1078382 1078516 r 1 0910 k k k k k ie e rpmH_bact: 5 = Translation ribosomal protein Equivalog L34 280204 281721 r 3 0218 d k k d k n e glycerol_kin: 5 = Lipid salvage and glycerol kinase Equivalog biogenesis 344859 345422 f 3 0271 k k k d k e ie T6A_YeaZ: tRNA 5 = tRNA threonylcarbamoyl Equivalog modification adenosine modification protein YeaZ 176449 176562 r 2 0135 j j j j j x x x 6 = not a x real gene 746854 746941 x x 0603 j j j j j x x x, not Small 6 = not a x nucleolar RNA real gene snR69 958511 958648 f 8 0815 j j j j j x x x 6 = not a x real gene 76906 77202 r 1 0051 j j j d k x x x 6 = not a x real gene 27639 28301 r 1 0918 j j j j j x x imidazoleglycerol- 7 = plasmid phosphate plasmid dehydratase 29270 31258 f 1 0913 j j j j j x x tetracycline 7 = plasmid resistance protein plasmid TetM 637296 637404 r 6 0532 i r d r r e e 5S rRNA 8 = RNA deleted 637479 640373 r 6 0533 i r d r r e e 23S rRNA 8 = RNA deleted 640604 642127 r 6 0534 i r d r r e e 16S rRNA 8 = RNA deleted 18716 20302 f 1 0013 k k d k k n n mycoides cluster 8 = Lipoprotein lipoprotein, deleted LppA/P72 family 44601 44810 r 1 0028 k k d k k n n cspB, RNA/DNA 8 = Unclear chaperone deleted 74945 75412 r 1 0048 k k d k k n n cytidine and 8 = Nucleotide deoxycytidylate deleted salvage deaminase zinc- binding region 87981 88295 f 1 0062 k k d k k n n PF08921 domain 8 = Unclear protein deleted 130730 131305 f 1 0096 d k d k k n n YigZ family protein 8 = Proteolysis deleted 334375 335094 r 3 0258 d k d k k n n KR domain protein 8 = Proteolysis deleted 357015 359429 r 3 0278 d k d k k n n phosphoenolpyruvate- 8 = Carbon source dependent sugar deleted transport & PTS family porter, catabolism EIIA 2 component 359688 359975 f 3 0279 d k d k k n n hypothetical protein 8 = Unclear deleted 363145 364032 f 3 0284 k k d k k n n hypothetical protein 8 = Unclear deleted 421586 422005 f 4 0333 k k d k k n n hypothetical protein 8 = Lipoprotein deleted 422445 422924 f 4 0334 k k d k k n n lipoprotein, putative 8 = Lipoprotein deleted 423315 423773 f 4 0335 k k d k k n n lipoprotein, putative 8 = Lipoprotein deleted 423939 424388 f 4 0336 k k d k k n n phosphoenolpyruvate- 8 = Transport dependent sugar deleted PTS family porter, EIIA 2 component 441952 442461 r 4 0351 d k d k k n n recombination 8 = DNA repair protein U deleted 444649 445872 f 4 0355 d k d k k n n PF03382 family 8 = Lipoprotein protein deleted 458292 458597 f 4 0370 k k d k k n n single-strand 8 = DNA replication binding family deleted protein 551789 552253 f 5 0446 k k d k k n n hypothetical protein 8 = Unclear deleted 614766 615842 r 5 0514 d k d k k n n hypothetical protein 8 = Unclear deleted 812217 813128 f 7 0677 d k d k k n n membrane protein, 8 = Unclear putative deleted 975825 976622 f 8 0829 d k d k k n n putative 8 = Unclear deoxyribonuclease deleted YcfH 1074592 1074750 r 1 0905 k k d k k n n ATPase, AAA 8 = Unclear domain protein deleted 594474 595394 f 5 0487 k d d k k i _(—) unknown 8 = Unclear deleted 595403 596506 f 5 0488 d d d k k i _(—) GTPase_YqeH: 8 = Ribosome ribosome biogenesis deleted biogenesis GTPase YqeH, bsub yqeH is essential 25111 25215 f 1 0916 j j d j j x x RNAI 8 = plasmid deleted 25426 26286 r 1 0914 j j d j j x x beta-lactamase 8 = plasmid deleted 26357 27529 r 1 0917 j j d j j x x transposase, mutator 8 = plasmid family deleted 31535 34618 f 1 0915 j j d j j x x beta-galactosidase 8 = plasmid deleted 46716 47582 r 1 0031 d k d d k n n Hsp33, targetted EF- 8 = Proteolysis Tu degradation deleted 59775 60968 f 1 0035 d k d d k n n PF03382 family 8 = Lipoprotein protein deleted 61277 62263 f 1 0036 k k d d k n n putative D-lactate 8 = Metabolic dehydrogenase deleted process 62282 63406 f 1 0037 d k d d k n n transporter, auxin 8 = Efflux efflux carrier deleted domain protein 63762 65045 f 1 0038 d k d d k n n AAA domain 8 = Unclear protein deleted 107089 108219 r 1 0078 k k d d k n n alpha/beta hydrolase 8 = Unclear family protein deleted 279390 280163 r 3 0217 d k d d k n n transporter, major 8 = Lipid salvage and intrinsic protein deleted biogenesis (MIP) family protein 281738 282901 r 3 0219 d k d d k n n FAD dependent 8 = Lipid salvage and oxidoreductase deleted biogenesis 521827 522282 r 5 0417 k k d d d n n PF13274 family 8 = Mobile element protein deleted & DNA restriction 538691 539344 f 5 0436 k k d d d n n ung: uracil-DNA 8 = DNA repair glycosylase deleted 583080 584237 r 5 0476 d k d d d n n nagA: N- 8 = Carbon source acetylglucosamine- deleted transport & 6-phosphate catabolism deacetylase 584406 585140 f 5 0477 d k d d k n n hypothetical protein 8 = Unclear deleted 587539 589539 f 5 0480 d k d d d n n hypothetical protein 8 = Unclear deleted 604970 605419 r 5 0496 k k d d d n n YhcH/YjgK/YiaL 8 = Unclear family protein deleted 605428 607131 r 5 0497 d k d d d n n transporter, SSS 8 = Transport family deleted 607133 608020 r 5 0498 d k d d d n n N-acetylneuraminate 8 = Carbon source lyase deleted transport & catabolism 20317 21831 f 1 0014 k d d d d n _(—) mycoides cluster 8 = Lipoprotein lipoprotein, deleted LppA/P72 family 21864 23393 f 1 0015 k d d d d n _(—) mycoides cluster 8 = Lipoprotein lipoprotein, deleted LppA/P72 family 23411 25009 f 1 0016 k d d d d n _(—) mycoides cluster 8 = Lipoprotein lipoprotein, deleted LppA/P72 family 35234 35776 f 1 0921 d d d d d n _(—) transposase 8 = Mobile element deleted & DNA restriction 35914 36645 f 1 0922 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 36687 37673 r 1 0019 d d d d d n _(—) mannitol 8 = Carbon source dehydrogenase C- deleted transport & terminal domain catabolism protein 37674 38471 r 1 0020 d d d d d n _(—) transcriptional 8 = Regulation regulator, RpiR deleted family 38505 38921 r 1 0021 d d d d d n _(—) phosphoenolpyruvate- 8 = Carbon source dependent sugar deleted transport & PTS family porter, catabolism EIIA 2 component 38921 39679 r 1 0022 d d d d d n _(—) sorbitol-6-phosphate 8 = Carbon source 2-dehydrogenase deleted transport & catabolism 39679 41238 r 1 0023 d d d d d n _(—) PTS system EIIC 8 = Carbon source component deleted transport & catabolism 41509 42891 r 1 0024 d d d d d n _(—) divergent AAA 8 = Unclear domain protein deleted 47674 50835 r 1 0032 d d d d d n _(—) GnsA/GnsB family 8 = Regulation protein deleted 67265 68110 f 1 0041 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 75529 76491 r 1 0050 k d d d d n _(—) guanosine 8 = Nucleotide monophosphate deleted salvage reductase 77327 77647 r 1 0052 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 77755 79851 r 1 0053 d d d d d n _(—) putative peptidase 8 = Lipoprotein deleted 80688 81977 r 1 0055 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 81979 82818 r 1 0056 d d d d d n _(—) DNA adenine 8 = Mobile element methylase deleted & DNA restriction 83287 83577 f 1 0057 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 83596 84177 f 1 0058 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 84292 85410 f 1 0059 d d d d d n _(—) alaDH: alanine 8 = Unclear dehydrogenase deleted 97603 99669 f 1 0072 d d d d d n _(—) beta-lactamase 8 = Unclear family protein deleted 99702 102434 r 1 0073 d d d d d n _(—) PhnE: phosphonate 8 = Transport ABC transporter, deleted permease protein PhnE 102438 103190 r 1 0074 k d d d d n _(—) ABC_phnC: 8 = Transport phosphonate ABC deleted transporter, ATP- binding protein 103204 104574 r 1 0075 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 111503 112138 r 1 0083 k d d d d in _(—) hypothetical protein 8 = Unclear deleted 112555 113967 r 1 0084 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 114118 116367 r 1 0085 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 116412 117539 r 1 0086 d d d d d n _(—) hypothetical protein 8 = Lipoprotein deleted 118095 118508 r 1 0087 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 118767 120617 r 1 0088 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 120876 122567 r 1 0089 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 122730 123332 r 1 0923 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 123560 126049 r 1 0092 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 126220 126990 r 1 0093 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 132495 133511 f 1 0098 d d d d d n _(—) CCATC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 133501 134418 f 1 0099 d d d d d n _(—) CCATC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 134396 135979 f 1 0100 d d d d d n _(—) CCATC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 136419 137714 f 2 0101 d d d d d n _(—) AAA domain 8 = Unclear protein deleted 138374 138649 f 2 0102 k d d d d in _(—) hypothetical protein 8 = Unclear deleted 138651 139208 f 2 0103 k d d d d n _(—) ytaG 8 = Cofactor deleted transport and salvage 139210 140016 r 2 0104 k d d d d n _(—) ribosomal RNA 8 = rRNA large subunit deleted modification methyltransferase J 144042 144794 r 2 0110 k d d d d n _(—) riboflavin 8 = Cofactor kinase/FAD deleted transport and synthetase salvage 144869 144944 r 2 0111 r d d d d n _(—) tRNA-Lys 8 = RNA deleted 145008 147287 r 2 0112 d d d d d n _(—) peptidase, S41 8 = Proteolysis family deleted 152703 154241 f 2 0118 d d d d d n _(—) ccmA: heme ABC 8 = Transport exporter, ATP- deleted binding protein CcmA 154271 155068 r 2 0119 d d d d d n _(—) hydrolase, TatD 8 = Unclear family deleted 155264 156490 f 2 0120 d d d d d n _(—) threonine ammonia- 8 = Unclear lyase deleted 156705 158285 f 2 0121 d d d d d n _(—) membrane protein, 8 = Unclear putative deleted 158297 159157 f 2 0122 d d d d d n _(—) alpha/beta hydrolase 8 = Unclear family protein deleted 159440 160150 f 2 0123 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 160552 161883 f 2 0124 d d d d d n _(—) membrane protein, 8 = Carbon source putative deleted transport & catabolism 162231 163934 f 2 0125 d d d d d n _(—) PTS system EIIC 8 = Carbon source component deleted transport & catabolism 169010 169984 f 2 0130 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 174619 176106 r 2 0134 d d d d d n _(—) transporter, major 8 = Transport facilitator family deleted protein 176702 177406 f 2 0136 d d d d d n _(—) glycerophosphodiester 8 = Lipid salvage and phosphodiesterase deleted biogenesis family protein 193471 195336 f 2 0152 d d d d d in _(—) putative PTS system 8 = Transport IIBC component deleted 195320 197590 f 2 0153 d d d d d n _(—) glycoside hydrolase, 8 = Carbon source family 31 deleted transport & catabolism 200904 201506 f 2 0155 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 201664 202329 r 2 0156 k d d d d n _(—) tRNA (guanine-N(7)-)- 8 = tRNA methyltransferase deleted modification 202331 203734 r 2 0157 d d d d d n _(—) mgtE: magnesium 8 = Transport transporter deleted 204414 206543 f 2 0159 d d d d d n _(—) transglutaminase- 8 = Lipoprotein like protein deleted 206563 207315 r 2 0160 d d d d d n _(—) nucleotidyl 8 = Unclear transferase, PF08843 deleted family 207402 207998 r 2 0161 d d d d d n _(—) PF13338 domain 8 = Unclear protein deleted 208209 209415 r 2 0162 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 222395 223690 r 2 0170 d d d d d n _(—) AAA domain 8 = Unclear protein deleted 223785 225089 r 2 0171 d d d d d n _(—) AAA domain 8 = Unclear protein deleted 225321 226202 f 2 0172 d d d d d n _(—) Cof-like hydrolase 8 = Unclear deleted 226236 227933 r 2 0173 k d d d d n _(—) mycoides cluster 8 = Lipoprotein lipoprotein, deleted LppA/P72 family 228150 228296 f 2 0174 d d d d d n _(—) haloacid 8 = Unclear dehalogenase-like deleted hydrolase domain protein 228333 230015 r 2 0175 d d d d d n _(—) mycoides cluster 8 = Lipoprotein lipoprotein, deleted LppA/P72 family 230125 230754 r 2 0176 k d d d d n _(—) beta- 8 = Carbon source phosphoglucomutase deleted transport & catabolism 230754 232553 r 2 0177 d d d d d n _(—) neopullulanase 8 = Unclear deleted 232672 234468 r 2 0178 d d d d d n _(—) neopullulanase 8 = Unclear deleted 234726 236525 f 2 0179 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 236820 238610 f 2 0180 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 238692 239183 f 2 0181 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 239198 241615 f 2 0182 d d d d d n _(—) ABC transporter, 8 = Carbon source permease protein deleted transport & catabolism 241630 244170 f 2 0183 d d d d d n _(—) ABC transporter, 8 = Carbon source permease protein deleted transport & catabolism 244172 245257 f 2 0184 d d d d d n _(—) putative 8 = Carbon source spermidine/putrescine deleted transport & ABC transporter, catabolism ATP-binding protein PotA 245365 247662 f 2 0185 d d d d d in _(—) glycoside hydrolase, 8 = Carbon source family 65, central deleted transport & catalytic domain catabolism protein 247664 249268 f 2 0186 d d d d d n _(—) putative glucan 1,6- 8 = Carbon source alpha-glucosidase deleted transport & catabolism 249291 250010 f 2 0187 k d d d d n _(—) UbiC transcription 8 = Regulation regulator-associated deleted domain protein 250039 251832 r 2 0188 d d d d d n _(—) pepF: 8 = Proteolysis oligoendopeptidase deleted F 251832 252515 r 2 0189 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 252531 253886 r 2 0190 d d d d d n _(—) cytosol 8 = Proteolysis aminopeptidase deleted family, catalytic domain protein 253998 254180 r 2 0191 d d d d d e _(—) hypothetical protein 8 = Unclear deleted 254459 255079 f 2 0192 d d d d d n _(—) chromate transport 8 = Transport protein deleted 255079 255756 f 2 0193 d d d d d n _(—) chromate transport 8 = Transport protein deleted 255890 256849 f 2 0194 d d d d d n _(—) Abi-like protein 8 = Unclear deleted 266177 267436 f 3 0204 k d d d d n _(—) rsmB: 16S rRNA 8 = rRNA (cytosine(967)- deleted modification C(5))- methyltransferase 267440 269269 r 3 0205 d d d d d n _(—) TypA_BipA: GTP- 8 = Translation binding protein deleted TypA/BipA 269514 270271 f 3 0206 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 270334 271125 f 3 0207 d d d d d n _(—) hypothetical protein 8 = Lipoprotein deleted 271254 272045 f 3 0208 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 272099 273016 r 3 0209 k d d d d n _(—) N-acetylmuramic 8 = Carbon source acid 6-phosphate deleted transport & etherase catabolism 273009 274706 r 3 0210 k d d d d n _(—) PTS system EIIC 8 = Carbon source component deleted transport & catabolism 274828 275661 r 3 0211 k d d d d n _(—) SIS domain protein 8 = Regulation deleted 275665 275868 r 3 0212 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 288390 289754 f 3 0223 d d d d d n _(—) putative NADH 8 = Redox oxidase deleted homeostasis 289757 290761 f 3 0224 d d d d d n _(—) lipoyltransferase and 8 = Metabolic lipoate-protein deleted process ligase 290805 291917 f 3 0225 d d d d d n _(—) PDH_E1_alph_x: 8 = Metabolic pyruvate deleted process dehydrogenase (acetyl-transferring) Ecomponent, alpha subunit 291917 292906 f 3 0226 d d d d d n _(—) K000162 pyruvate 8 = Metabolic dehydrogenase E1 deleted process component 298421 300376 f 3 0231 d d d d d n _(—) putative lipoprotein 8 = Lipoprotein deleted 300386 300808 f 3 0232 d d d d k n _(—) coaD_prev_kdtB: 8 = Cofactor pantetheine- deleted transport and phosphate salvage adenylyltransferase 303533 304159 f 3 0236 d d d d d n _(—) dha_L_ycgS: 8 = Metabolic dihydroxyacetone deleted process kinase, L subunit 304167 304562 f 3 0237 d d d d d n _(—) domain 8 = Metabolic deleted process 308601 311333 f 3 0241 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 311451 313136 f 3 0242 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 313147 315438 f 3 0243 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 315501 317768 f 3 0244 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 318242 320383 f 3 0245 d d d d k n _(—) PTS system EIIC 8 = Transport component domain deleted protein 320410 323316 r 3 0246 d d d d k in _(—) putative calcium- 8 = Transport translocating P-type deleted ATPase, PMCA- type 325823 326515 f 3 0251 d d d d k n _(—) hypothetical protein 8 = Unclear deleted 326516 327238 r 3 0252 d d d d k n _(—) NAD(P)H-binding 8 = Redox protein, PF13460 deleted homeostasis family 329643 330704 r 3 0255 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 330770 332392 r 3 0256 k d d d k n _(—) amino acid permease 8 = Transport deleted 338719 339345 r 3 0261 d d d d d n _(—) thiamine 8 = Cofactor diphosphokinase deleted transport and salvage 342126 342883 r 3 0268 d d d d d n _(—) phosphoprotein 8 = Unclear phosphatase deleted 342912 344321 r 3 0269 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 345458 350779 r 3 0272 d d d d d n _(—) efflux ABC 8 = Efflux transporter, deleted permease protein 350992 354132 f 3 0273 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 354147 354761 f 3 0274 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 354901 355422 f 3 0275 d d d d k n _(—) lipoprotein, putative 8 = Lipoprotein deleted 355682 356302 f 3 0276 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 356463 356975 f 3 0277 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 371714 373759 r 3 0292 d d d d d n _(—) peptidase, S41 8 = Proteolysis family deleted 373784 375754 r 3 0293 k d d d d n _(—) putative lipoprotein 8 = Lipoprotein deleted 389506 390282 r 4 0306 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 390558 390911 f 4 0307 k d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 392042 392911 r 4 0309 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 393024 393824 f 4 0310 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 393879 394955 r 4 0311 d d d d d n _(—) RmuC domain 8 = Unclear protein deleted 395171 397891 f 4 0312 d d d d d n _(—) papain family 8 = Unclear cysteine protease deleted 398248 399522 f 4 0313 k d d d d n _(—) putative 8 = tRNA tRNA: m(5)U-54 deleted modification methyltransferase 402993 404537 f 4 0318 d d d d d n _(—) SNF2 family N- 8 = Unclear terminal domain deleted protein 404627 407524 f 4 0319 d d d d d n _(—) transglutaminase- 8 = Lipoprotein like protein deleted 407532 408329 f 4 0320 d d d d d n _(—) Ndr family protein 8 = Acylglycerol deleted breakdown 408482 408910 f 4 0321 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 409235 410692 f 4 0322 d d d d d n _(—) divergent AAA 8 = Unclear domain protein deleted 410710 412203 r 4 0323 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 412372 412896 r 4 0324 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 419454 420167 f 4 0331 d d d d d n _(—) putative 8 = Regulation transcriptional deleted regulator, YeeN 424400 426208 f 4 0337 d d d d d n _(—) PTS system sugar- 8 = Transport specific permease deleted component 427501 429321 f 4 0339 k d d d d n _(—) Na+ ABC 8 = Transport transporter, ATP- deleted binding component 429337 431127 f 4 0340 k d d d d n _(—) membrane protein, 8 = Transport putative deleted 431156 432556 f 4 0341 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 432682 434868 f 4 0342 d d d d d n _(—) hypothetical protein 8 = Lipoprotein deleted 435005 435700 f 4 0343 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 440093 441082 f 4 0349 k d d d d n _(—) NAD-dependent 8 = Metabolic glycerol-3- deleted process phosphate dehydrogenase N- terminal 443061 444476 f 4 0354 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 446339 446809 f 4 0357 d d d d d n _(—) competence/damage- 8 = Unclear inducible protein deleted CinA 446852 447889 f 4 0358 d d d d d n _(—) recA: protein RecA 8 = DNA repair deleted 454449 455072 f 4 0367 d d d d d n _(—) ribonuclease HII 8 = DNA replication deleted 455094 456890 r 4 0368 d d d d d n _(—) rhodanese-like 8 = Unclear protein deleted 456893 458131 r 4 0369 d d d d d n _(—) sulfur transport 8 = Transport deleted 470394 471113 f 4 0383 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 471190 471927 f 4 0384 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 471937 472617 f 4 0385 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 472656 473501 f 4 0386 d d d d d in _(—) lipoprotein, putative 8 = Lipoprotein deleted 479541 480827 f 4 0393 d d d d d n _(—) tig: trigger factor 8 = Translation deleted 483409 484644 f 4 0395 d d d d d n _(—) MgsA AAA+ 8 = Unclear ATPase family deleted protein 484673 486898 r 4 0396 d d d d d n _(—) transglutaminase- 8 = Lipoprotein like protein deleted 486949 489240 r 4 0397 d d d d d n _(—) transglutaminase- 8 = Lipoprotein like protein deleted 553645 554187 f 5 0449 d d d d d n _(—) transposase 8 = Mobile element deleted & DNA restriction 554325 555056 f 5 0450 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 561557 563740 f 5 0454 k d d d d n _(—) putative helicase, 8 = DNA repair RecD/TraA family deleted 563782 565299 r 5 0455 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 565789 566184 f 5 0924 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 566641 570003 f 5 0460 d d d d d e _(—) PF03382 family 8 = Unclear protein deleted 570054 570785 r 5 0461 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 570923 571465 r 5 0462 d d d d d n _(—) transposase 8 = Mobile element deleted & DNA restriction 571530 572708 r 5 0463 d d d d d n _(—) oxidoreductase, 8 = Redox FAD/FMN deleted homeostasis dependent 572708 573745 r 5 0464 d d d d d n _(—) lipoyltransferase and 8 = Metabolic lipoate-protein deleted process ligase 573723 574580 r 5 0465 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 574582 574926 r 5 0466 k d d d d n _(—) glycine cleavage H- 8 = Unclear protein deleted 575113 575907 f 5 0467 d d d d d n _(—) alpha/beta hydrolase 8 = Acylglycerol family protein deleted breakdown 575909 576703 f 5 0468 d d d d d n _(—) alpha/beta hydrolase 8 = Acylglycerol family protein deleted breakdown 576697 577506 f 5 0469 d d d d d n _(—) PF05057 family 8 = Acylglycerol protein deleted breakdown 577525 578658 r 5 0470 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 578789 578920 r 5 0471 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 578922 580322 f 5 0472 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 580469 581188 f 5 0473 d d d d d n _(—) ABC transporter, 8 = Transport ATP-binding protein deleted 581188 581943 f 5 0474 k d d d d n _(—) ABC transporter, 8 = Transport ATP-binding protein deleted 590637 591197 f 5 0483 k d d d d n _(—) putative Holliday 8 = DNA repair junction DNA deleted helicase RuvA 591214 592137 f 5 0484 k d d d d n _(—) ruvB: Holliday 8 = DNA repair junction DNA deleted helicase RuvB 592215 593573 f 5 0485 d d d d d n _(—) pyridine nucleotide- 8 = Unclear disulfide deleted oxidoreductase 593589 594398 r 5 0486 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 596515 597738 r 5 0489 d d d d d n _(—) ImpB/MucB/SamB 8 = DNA repair family protein deleted 597882 600620 f 5 0490 d d d d d n _(—) papain family 8 = Proteolysis cysteine protease deleted 600698 601321 r 5 0491 k d d d d n _(—) udk: uridine kinase 8 = Nucleotide deleted salvage 601345 601785 r 5 0492 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 622614 623603 r 6 0520 d d d d d in _(—) alpha/beta hydrolase 8 = Unclear family protein deleted 623616 624032 r 6 0521 k d d d d n _(—) PF04472 family 8 = Unclear protein deleted 624044 625201 r 6 0522 k d d d d n _(—) ftsZ: cell division 8 = Cell division protein FtsZ deleted 628122 628640 r 6 0527 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 645411 646016 r 6 0538 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 654952 655188 r 6 0546 k d d d d n _(—) sucrose-6F- 8 = Carbon source phosphate deleted transport & phosphohydrolase catabolism domain protein 655176 655817 r 6 0547 k d d d d n _(—) HAD hydrolase, 8 = Unclear family IIB deleted 655817 656362 r 6 0548 d d d d d n _(—) putative tRNA 8 = rRNA (cytidine(34)-2′-O)- deleted modification methyltransferase 656375 656977 r 6 0549 k d d d d n _(—) non-canonical 8 = Nucleotide purine NTP deleted salvage pyrophosphatase, RdgB/HAMfamily 657179 659694 f 6 0550 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 659874 662378 f 6 0551 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 662695 663483 f 6 0552 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 663752 664993 f 6 0553 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 665166 665999 f 6 0554 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 666028 668043 f 6 0555 d d d d d n _(—) type IV secretory 8 = Mobile element system Conjugative deleted & DNA DNA transfer restriction 668059 669015 f 6 0556 d d d d d n _(—) PF03382 family 8 = Mobile element protein deleted & DNA restriction 668999 669916 f 6 0557 d d d d d n _(—) PF03382 family 8 = Mobile element protein deleted & DNA restriction 669931 670347 f 6 0558 k d d d d n _(—) single-strand 8 = Mobile element binding family deleted & DNA protein restriction 670612 671802 f 6 0559 d d d d d n _(—) PF06114 domain 8 = Mobile element protein deleted & DNA restriction 672040 673572 f 6 0560 d d d d d n _(—) lipoprotein, putative 8 = Mobile element deleted & DNA restriction 673574 673765 f 6 0561 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 673862 674092 f 6 0562 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 674120 676843 f 6 0563 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 676950 677135 f 6 0564 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 677225 677602 f 6 0565 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 677609 679531 f 6 0566 d d d d d n _(—) membrane protein, 8 = Mobile element putative deleted & DNA restriction 679556 682399 f 6 0567 d d d d d n _(—) AAA-like domain 8 = Mobile element protein deleted & DNA restriction 682412 683077 f 6 0568 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 683124 688091 f 6 0569 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 688547 689293 f 6 0570 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 689489 689854 f 6 0571 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 690078 690992 f 6 0572 d d d d d n _(—) CobQ/CobB/MinD/ 8 = Mobile element ParA nucleotide deleted & DNA binding domain restriction protein 691042 692274 f 6 0573 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 692861 695401 f 6 0574 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 695457 696836 r 6 0575 d d d d d n _(—) ATP synthase 8 = Mobile element alpha/beta chain, C- deleted & DNA terminal domain restriction 696836 698383 r 6 0576 d d d d d n _(—) putative ATP 8 = Mobile element synthase F1, alpha deleted & DNA subunit restriction 698388 700691 r 6 0577 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 700693 701136 r 6 0578 k d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 701136 702047 r 6 0579 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 702049 702543 r 6 0580 d d d d d n _(—) PF10896 family 8 = Mobile element protein deleted & DNA restriction 702518 703981 r 6 0581 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 704013 706598 r 6 0582 d d d d d n _(—) putative peptidase 8 = Mobile element deleted & DNA restriction 706621 708873 r 6 0583 d d d d d n _(—) mycoplasma 8 = Mobile element virulence signal deleted & DNA region restriction (Myco_arth_vir_N) 709145 711715 r 6 0584 d d d d d n _(—) putative peptidase 8 = Mobile element deleted & DNA restriction 711735 713984 r 6 0585 d d d d d n _(—) mycoplasma 8 =deleted Mobile element virulence signal & DNA region restriction (Myco_arth_vir_N) 714177 716732 r 6 0586 d d d d d n _(—) putative peptidase 8 = Proteolysis deleted 716747 718999 r 6 0587 d d d d d n _(—) mycoplasma 8 = Mobile element virulence signal deleted & DNA region restriction (Myco_arth_vir_N) 719219 721780 r 6 0588 d d d d d n _(—) putative peptidase 8 = Proteolysis deleted 721800 724058 r 6 0589 d d d d d n _(—) mycoplasma 8 = Mobile element virulence signal deleted & DNA region restriction (Myco_arth_vir_N) 724657 725553 r 6 0925 d d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 725660 726226 r 6 0926 k d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 726177 726560 f 6 0927 k d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 726553 727071 r 6 0590 d d d d d n _(—) type III restriction 8 = Mobile element enzyme, res subunit deleted & DNA restriction 727058 728260 r 6 0591 d d d d d n _(—) DNA methylase 8 = Mobile element family protein deleted & DNA restriction 729115 730587 r 6 0592 d d d d d n _(—) PF09903 family 8 = Unclear protein deleted 730811 731089 r 6 0593 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 731220 732998 r 6 0594 d d d d d n _(—) MATE domain 8 = Efflux protein deleted 733270 734646 f 6 0595 d d d d d n _(—) type III restriction 8 = Mobile element enzyme, res subunit deleted & DNA restriction 735282 737948 f 6 0596 d d d d d n _(—) AAA domain 8 = Unclear protein deleted 738368 738793 f 6 0597 d d d d d n _(—) Fic/DOC family 8 = Cell division protein deleted 739136 740662 f 6 0598 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 744738 746000 f 6 0602 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 746503 747171 f 6 0604 d d d d d n _(—) LemA family 8 = Unclear protein deleted 747354 747662 f 6 0605 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 752661 753485 r 6 0610 d d d d d n _(—) fpg: DNA- 8 = DNA repair formamidopyrimidine deleted glycosylase 765083 765445 r 6 0622 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 765432 766502 r 6 0623 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 766799 767731 r 6 0625 d d d d d n _(—) putative carbamate 8 = Carbon source kinase deleted transport & catabolism 767848 768942 r 6 0626 d d d d d n _(—) aguA, agmatine 8 = Carbon source deiminase, agmatine deleted transport & to putrescine via N- catabolism carbamoylputrescine 768949 770367 r 6 0627 d d d d d n _(—) amino acid permease 8 = Transport deleted 770394 771491 r 6 0628 d d d d d n _(—) ornithine 8 = Carbon source carbamoyltransferase deleted transport & catabolism 771786 772163 r 6 0629 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 772384 773271 f 6 0630 d d d d d n _(—) ABC transporter, 8 = Transport ATP-binding protein deleted 773273 774037 f 6 0631 d d d d d n _(—) ABC-2 family 8 = Transport transporter protein deleted 774638 776197 r 6 0633 d d d d d n _(—) membrane protein, 8 = Unclear putative deleted 806351 807349 r 6 0673 d d d d d n _(—) dhaK1: 8 = Metabolic dihydroxyacetone deleted process kinase, DhaK subunit 807457 809085 r 6 0674 d d d d d n _(—) putative alpha, alpha- 8 = Carbon source phosphotrehalase deleted transport & catabolism 809093 810640 r 6 0675 d d d d d n _(—) PTS system EIIC 8 = Carbon source component deleted transport & catabolism 810706 811692 r 6 0676 d d d d d n _(—) transcriptional 8 = Regulation regulator, LacI deleted family 813801 814964 r 7 0682 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 815077 815832 r 7 0683 d d d d d n _(—) tetraspanin family 8 = Unclear protein deleted 832678 833904 r 7 0698 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 833993 834922 r 7 0699 d d d d d n _(—) peptide-methionine 8 = Redox (S)-S-oxide deleted homeostasis reductase MsrA/ methionine-R- sulfoxide reductase MsrB multi-domain protein 835036 837222 r 7 0700 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 837470 839818 r 7 0701 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 839827 840729 r 7 0702 d d d d d n _(—) membrane protein, 8 = Unclear putative deleted 840856 842691 r 7 0703 d d d d d n _(—) PF03235 family 8 = Unclear protein deleted 842694 843260 r 7 0704 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 843263 844099 r 7 0705 k d d d d n _(—) hypothetical protein 8 = Mobile element deleted & DNA restriction 849525 850715 f 7 0711 d d d d d n _(—) aminotransferase, 8 = Unclear class I/II deleted 850725 851126 f 7 0712 d d d d d n _(—) putative 8 = Unclear endoribonuclease L- deleted PSP domain protein 851162 852493 r 7 0713 d d d d d n _(—) PF03382 family 8 = Unclear protein deleted 852523 853779 r 7 0714 d d d d d n _(—) PF03382 family 8 = Unclear protein deleted 853966 855234 r 7 0715 d d d d d n _(—) PF03382 family 8 = Unclear protein deleted 855376 856518 f 7 0716 d d d d d n _(—) alpha/beta hydrolase 8 = Unclear family protein deleted 862389 863402 f 7 0731 d d d d d n _(—) nucleotidyl 8 = Unclear transferase, PF08843 deleted family 865972 867285 f 7 0734 d d d d d n _(—) putative pyrimidine- 8 = Nucleotide nucleoside deleted salvage phosphorylase 867340 868071 r 7 0735 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 868209 868751 r 7 0736 d d d d d n _(—) transposase 8 = Mobile element deleted & DNA restriction 868816 869103 r 7 0737 d d d d d n _(—) hypothetical protein 8 = Carbon source deleted transport & catabolism 869142 869264 r 7 0738 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 869269 869403 r 7 0739 d d d d d n _(—) hypothetical protein 8 = Carbon source deleted transport & catabolism 869453 869608 r 7 0740 k d d d d n? _(—) hypothetical protein 8 = Unclear deleted 869727 870719 r 7 0741 d d d d d n _(—) CCTTC-recognizing 8 = Mobile element Type II restriction deleted & DNA modification system restriction (MmyCII) endonuclease subunit 870721 873225 r 7 0742 d d d d d n _(—) CCTTC-recognizing 8 = Mobile element Type II restriction deleted & DNA modification system restriction (MmyCII) adenine/cytosine DNA methyltransferase subunit 873360 875396 r 7 0743 d d d d d n _(—) phosphoenolpyruvate- 8 = Carbon source dependent sugar deleted transport & PTS family porter, catabolism EIIA 2 component 875399 876334 r 7 0744 d d d d d n _(—) putative 1- 8 = Carbon source phosphofructokinase deleted transport & catabolism 876334 877032 r 7 0745 d d d d d n _(—) transcriptional 8 = Regulation regulator, DeoR deleted family 877185 878165 f 7 0746 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 879069 879533 r 7 0748 d d d d d n _(—) DNA-binding helix- 8 = Mobile element turn-helix protein deleted & DNA restriction 879588 880493 r 7 0749 d d d d d n _(—) putative 8 = Unclear lysophospholipase deleted 880596 883649 r 7 0750 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 883674 886715 r 7 0751 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 887085 887657 f 7 0752 d d d d d n _(—) Fic/DOC family 8 = Cell division protein deleted 887865 888137 f 7 0753 d d d d d n _(—) addiction module 8 = Unclear antitoxin, RelB/DinJ deleted family 888176 889081 r 7 0754 d d d d d n _(—) GCATC- 8 = Mobile element recognizing Type II deleted & DNA methyltransferase restriction 889074 890162 r 7 0755 d d d d d n _(—) GCATC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 890279 892318 f 7 0756 d d d d d n _(—) GCATC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 892356 893441 r 7 0757 d d d d d n _(—) PF05913 family 8 = Unclear protein deleted 893443 894831 r 7 0758 d d d d d n _(—) glycoside hydrolase, 8 = Carbon source family 1 deleted transport & catabolism 894824 895702 r 7 0759 k d d d d n _(—) ROK family protein 8 = Regulation deleted 895704 896162 r 7 0760 k d d d d n _(—) phosphoenolpyruvate- 8 = Transport dependent sugar deleted PTS family porter, EIIA 2 component 896176 898842 r 7 0761 d d d d d n _(—) glycoside hydrolase, 8 = Carbon source family 38, N- deleted transport & terminal domain catabolism protein 898854 900695 r 7 0762 d d d d d n _(—) PTS system EIIC 8 = Carbon source component deleted transport & catabolism 900710 901486 r 7 0763 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 901652 902203 r 7 0764 k d d d d n _(—) HGPRTase: 8 = Nucleotide hypoxanthine deleted salvage phosphoribosyltransferase 902239 903537 r 7 0765 d d d d d n _(—) purB: 8 = Nucleotide adenylosuccinate deleted salvage lyase 903530 904828 r 7 0766 k d d d d n _(—) purA: 8 = Nucleotide adenylosuccinate deleted salvage synthase 905049 905201 r 7 0767 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 905182 905697 r 7 0768 d d d d d n _(—) GANTC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 905859 906578 r 7 0769 d d d d d n _(—) GANTC- 8 = Mobile element recognizing deleted & DNA TypeIIrestric- restriction tionmodification 906639 907484 r 7 0770 d d d d d n _(—) PF03382 family 8 = Unclear protein deleted 917651 918193 f 7 0780 d d d d d n _(—) transposase 8 = Mobile element deleted & DNA restriction 918331 919062 f 7 0781 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 919157 920284 f 7 0782 d d d d d n _(—) beta-lactamase 8 = Unclear deleted 920380 921345 f 7 0783 d d d d d n _(—) ROK family protein 8 = Regulation deleted 921413 922345 r 7 0784 d d d d d n _(—) putative carbamate 8 = Carbon source kinase deleted transport & catabolism 922358 923821 r 7 0785 k d d d d n _(—) C4-dicarboxylate 8 = Carbon source anaerobic carrier deleted transport & catabolism 923900 924895 r 7 0786 d d d d d n _(—) orni_carb_tr: 8 =deleted Carbon source ornithine transport & carbamoyltransferase catabolism 928331 929518 f 7 0788 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 939085 939381 f 8 0801 d d d d d n _(—) rhodanese-like 8 = Unclear protein deleted 939386 941581 r 8 0802 d d d d d n _(—) putative peptidase 8 = Lipoprotein deleted 953142 954020 r 8 0811 d d d d d n _(—) galU 8 = Unclear deleted 954550 955926 r 8 0812 d d d d d n _(—) EpsG 8 = Lipid salvage and deleted biogenesis 958936 959442 r 8 0928 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 959417 959686 f 8 0929 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 959695 960048 f 8 0816 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 987122 993043 f 8 0841 k d d d d n _(—) hypothetical protein 8 = Unclear deleted 993053 996010 f 8 0842 k d d d d n _(—) ABC transporter, 8 = Lipoprotein substrate-binding deleted protein, family 5 996010 997083 f 8 0843 k d d d d n _(—) ABC transporter, 8 = Transport permease protein deleted 997085 998095 f 8 0844 k d d d d n _(—) ABC transporter, 8 = Transport permease protein deleted 998103 999548 f 8 0845 k d d d d n _(—) oligopeptide/dipeptide 8 = Transport transporter, C- deleted terminal domain protein 999541 1000863 f 8 0846 k d d d d n _(—) ABC transporter, 8 = Transport ATP-binding protein deleted 1000900 1001631 r 8 0267 d d d d d n _(—) integrase core 8 = Mobile element domain protein deleted & DNA restriction 1001769 1002311 r 8 0265 d d d d d n _(—) transposase 8 = Mobile element deleted & DNA restriction 1002392 1002898 r 8 0849 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 1003095 1003970 r 8 0850 d d d d d n _(—) PF03382 family 8 = Unclear protein deleted 1005324 1005833 r 8 0854 d d d d d i _(—) dual specificity 8 = Unclear phosphatase, deleted catalytic domain protein 1005833 1007533 r 8 0855 d d d d d n _(—) PTS system EIIC 8 = Transport component deleted 1007610 1009322 r 8 0856 d d d d d n _(—) PTS system EIIC 8 = Transport component deleted 1009630 1011018 f 8 0858 d d d d d n _(—) divergent AAA 8 = Regulation domain protein deleted 1011005 1011820 r 8 0857 d d d d d n _(—) SIS domain protein 8 = Unclear deleted 1014116 1014841 r 8 0860 k d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 1015242 1016126 r 8 0861 d d d d d n _(—) aromatic cluster 8 = Unclear surface protein deleted 1016113 1016943 r 8 0862 k d d d d n _(—) aromatic cluster 8 = Unclear surface protein deleted 1016930 1018279 r 8 0863 d d d d d n _(—) aromatic cluster 8 = Lipoprotein surface protein deleted 1018257 1018961 r 8 0864 d d d d d n _(—) bacitracin ABC 8 = Transport transporter, ATP- deleted binding protein BcrA family protein 1018963 1020834 r 8 0865 d d d d d n _(—) ABC-2 family 8 = Transport transporter protein deleted 1020973 1021860 r 8 0866 d d d d d n _(—) aromatic cluster 8 = Lipoprotein surface protein deleted 1021862 1022692 r 8 0867 k d d d d n _(—) aromatic cluster 8 = Unclear surface protein deleted 1022679 1023872 r 8 0868 k d d d d n _(—) aromatic cluster 8 = Lipoprotein surface protein deleted 1023891 1025786 r 8 0869 k d d d d n _(—) membrane protein, 8 = Transport putative deleted 1027731 1028438 r 8 0871 k d d d d n _(—) PF01863 family 8 = Proteolysis protein deleted 1037851 1038822 r 8 0880 d d d d d n _(—) DNA-binding 8 = Unclear protein HU deleted 1040770 1041414 r 8 0882 d d d d d in _(—) hypothetical protein 8 = Unclear deleted 1041471 1042451 r 8 0883 d d d d d n _(—) putative aspartate- 8 = Metabolic ammonia ligase deleted process 1042655 1043413 f 8 0884 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1048868 1049368 f 8 0888 k d d d d n _(—) isochorismatase 8 = Unclear family protein deleted 1049414 1050091 r 8 0889 d d d d d n _(—) nucleotidyl 8 = Unclear transferase, PF08843 deleted family 1050248 1050847 r 8 0890 d d d d d n _(—) PF13338 domain 8 = Unclear protein deleted 1051424 1052758 f 8 0891 d d d d d n _(—) PF03382 family 8 = Lipoprotein protein deleted 1053008 1054231 f 8 0892 d d d d d n _(—) PAP2 family protein 8 = Unclear deleted 1054362 1055567 f 8 0893 d d d d d n _(—) PAP2 domain 8 = Unclear protein deleted 1055650 1057914 r 8 0894 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1057924 1060185 r 8 0895 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1060502 1062757 r 8 0896 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1062767 1065028 r 8 0897 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1065223 1067475 r 8 0898 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1067499 1069739 r 8 0899 d d d d d n _(—) hypothetical protein 8 = Unclear deleted 1070055 1070690 f 8 0900 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 1070923 1071534 f 8 0901 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 1071824 1072303 f 8 0902 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 1072804 1073379 f 8 0903 d d d d d n _(—) lipoprotein, putative 8 = Lipoprotein deleted 1073640 1074323 f 8 0904 d d d d d n _(—) hypothetical protein 8 = Unclear deleted

In the course of making the HMG design, the following set of deletion rules were developed and used in the subsequent examples.

(1) Generally the entire coding region of each gene considered non-essential was deleted, including start and stop codons. (See exceptions below)

(2) When a cluster of more than one consecutive gene was deleted, the intergenic regions within the cluster were deleted also.

(3) Intergenic regions that flank a deleted gene, or a consecutive cluster of deleted genes, were retained.

(4) Parts of genes to be deleted were retained if they overlapped a retained gene.

(5) Parts of genes to be deleted were retained if they contained a ribosome binding site or promoter for a retained gene.

(6) When two genes were divergently transcribed, it was assumed the intergenic region separating them contained promoters for transcription in both directions.

(7) When a deletion resulted in converging transcripts a bidirectional terminator was inserted, if one was not already present.

Each of the HMG and Syn1.0 genomes was divided into 8 overlapping segments was chemically synthesized and assembled. Each of the HMG genomic fragments had a corresponding Syn1.0 genomic segment, which allowed untested pieces to be mixed-and-matched with viable Syn1.0 pieces in one-pot combinatorial assemblies or purposefully assembled in any specified combination. Additionally, each of the eight target segments (i.e., the HMG fragments) was moved into a ⅞^(th) Syn1.0 background by recombinase mediated cassette exchange (RMCE) (FIG. 10). Each of the eight target segments is referred to as a ⅛^(th) RGD, reduced genome design, in FIG. 10 and the Experimental Materials and Methods section. RMCE has been described in Noskov et al., Biol. Proced. Online. 17, 6 (2015), which is hereby incorporated by reference. Unique restriction sites (NotI) flanked each HMG or Syn1.0 segment in the resulting strains (FIGS. 2A2A-2C).

8 mycoplasma strains were produced as a result of moving each of the eight target segments into a ⅞^(th) Syn1.0 background by recombinase mediated cassette exchange (RMCE), each carrying one HMG segment. 8 other mycoplasma strains, each carrying one Syn1.0 segment, in each case flanked by NotI sites, were also produced. This facilitated the production of HMG and Syn1.0 segments because they could be recovered from bacterial cultures, which produced much higher yields of better quality DNA than yeast. All 8 HMG segments were tested in a Syn1.0 background, but only one of the segment designs produced viable colonies (HMG segment 2), and the cells grew poorly. As described in subsequent examples, more rigorous evaluations of dispensable genes were performed. There was also a need to repeatedly assess which remaining genes were dispensable as smaller and smaller genomes were produced (See FIG. 25).

In the HMG work described here, a semi-automated DNA synthesis procedure capable of rapidly generating error-free large DNA constructs starting from overlapping oligonucleotides was used. The procedure included (i) single-reaction assembly of 1.4-kb DNA fragments from overlapping oligonucleotides, (ii) eliminating synthesis errors and permitting single-round assembly and cloning of error-free 7-kb cassettes, (iii) cassette sequence verification to simultaneously identify hundreds of error-free clones in a single run, and (iv) rolling circle amplification (RCA) of large plasmid DNA derived from yeast. This procedure significantly increased the rate at which the Design-Build-Test cycle (DBT) was carried out. A non-limiting schematic illustration of the strategy for the DNA synthesis is shown in FIG. 21.

Example 2 Identification of Essential, Quasi-Essential, and Non-Essential Genes Using Tn5 Transposon Mutagenesis

This example shows the use of Tn5 transposon mutagenesis to identify essential, quasi-essential, and non-essential genes.

To obtain much better knowledge of which genes are essential versus non-essential, Tn5 transposon mutagenesis was performed (FIG. 3). An initial Tn5 disruption map was generated by transforming JCVI-Syn1.0 ΔRE ΔIS cells (Table 9) with an activated form of a 988-bp mini-Tn5 puromycin resistance transposon (FIG. 3). Transformed cells were selected on agar plates containing 10 μg/ml puromycin. Approximately 80,000 colonies, each arising from a single Tn5 insertion event, were pooled from the plates. A sample of DNA extracted from this P0 pool was mechanically sheared and analyzed for the sites of Tn5 insertion using inverse PCR and Miseq. The P0 data set contained ≈30,000 unique insertions. To remove slow growing mutagenized cells, a sample of the pooled P0 cells was serially passaged for more than 40 generations, and DNA was prepared and sequenced to generate a P4 data set containing ≈14,000 insertions (FIG. 4).

Genes were classified into 3 major groups according to the results of the Tn5 transposon mutagenesis: (1) genes that were not hit at all, or were sparsely hit in the terminal 20% of the 3′-end or the first few bases of the 5′-end were classified as essential “e” genes (also referred to as “e-genes”); (2) genes that were hit frequently by both P0 and P4 insertions were classified as non-essential “n” genes (also referred to as “n-genes”; and (3) genes hit primarily by P0 insertions but not P4 insertions were classified as quasi-essential, growth-impaired “i” genes (also referred to as “i-genes”). The use of transposon mutagenesis to identify nonessential genes has been described in Hutchison et al., Science 286:2165-9 (1999), which is hereby incorporated by reference. Cells with i-gene disruptions formed a continuum of growth impairment varying from minimal to severe. To highlight this growth continuum, i-genes with minimal growth disadvantage was designated as in-genes, and those with severe growth defect as “ie” genes. Of the 901 annotated protein and RNA coding genes in the Syn1.0 genome, 432 were initially classified as n-genes, 240 were e-genes, and 229 were i-genes (FIGS. 5, 22A-22B).

FIGS. 22A-22B show the three gene classifications based on Tn5 mutagenesis data. FIG. 22A shows a number of examples of the 3 gene classifications based on Tn5 mutagenesis data. The gene MMSYN1_0128 (the arrow starting at the right end of the top line) had P0 Tn5 inserts (open bars) and is a quasi-essential i-gene. The next gene (MMSYN1_0129) had no inserts and is an essential e-gene. The last gene (MMSYN1_0130) had both P0 (open bars) and P4 (black bars) inserts and is a non-essential n-gene. FIG. 22B shows the number of Syn1.0 genes in each Tn5 mutagenesis classification group. n-genes and in-genes were candidates for deletion in reduced genome designs.

When displayed on the Syn1.0 gene map (FIG. 6), P4 insertions hit the n-genes at high frequency whereas the e-genes had no hits, and i-genes were sparsely hit or not at all. The map shows that non-essential genes tended to occur in clusters far more often than expected by chance. Deletion analysis was used to confirm that most of the n-gene clusters could be deleted without loss of viability or significantly affecting growth rate (when displayed on the Syn1.0 gene map, FIG. 15). Individual gene clusters (or in some cases single genes) were replaced by the URA3 marker as follows. 50 bp sequences flanking the gene(s) to be deleted were added to the ends of the URA3 marker by PCR and the DNA was introduced into yeast cells carrying Syn1.0 genome. Yeast clones were selected on plates not containing uracil, confirmed by PCR, and transplanted to determine viability. Deletions fell into 3 classes: (1) Those resulting in no transplants, indicating deletion of an essential gene, (2) Those resulting in transplants with normal or near normal growth rates, indicating deletion of non-essential genes, and (3) Those resulting in transplants with slow growth, indicating deletion of quasi-essential, i-genes.

A large number of deletions, including all of the HMG deletions, were individually tested for viability and yielded valuable information for subsequent reduced genome designs. The transposon insertion data used in the HMG design was all collected from passage P0. Consequently genes with insertions included the genes subsequently characterized as quasi-essential i-genes, so some HMG deletions gave very small colonies, or were non-viable.

In addition to deleting individual clusters, step-wise scarless deletion (FIG. 23) of medium to large clusters was undertaken to produce a series of strains with progressively greater numbers of genes removed. Strain D22 with 255 genes and 357 kb of DNA removed grew at a rate similar to Syn1.0 (Table 9). These deletion studies verified that the set of deletion rules routinely yielded viable knockouts.

FIG. 23 is a schematic illustration showing the TREC deletion method. To generate a scarless deletion, the CORE cassette was PCR-amplified in two rounds to produce the knock-out cassette contained a 50 bp (“U” block) for homologous recombination, 50 bp (“D” block) repeated sequence, and a 50 bp (“D” block) for homologous recombination. Step 1, the cassette was transformed into a yeast strain harboring a mycoides genome and selected on SD minus plate. Correct target knock out was identified by PCR screening for insertion junctions (L and R). Step 2, galactose induction resulted in the expression of I-Sce I endonuclease, which cleaved the 18-bp I-Sce I site (open bar) to create a double-strand break that promoted homologous recombination between two tandem repeat sequences (“D” block). Step 3, recombination between two repeat sequences generated a scarless deletion. The deletion of a target region was confirmed by PCR using primers located up and down stream of the target region.

Step-Wise Genome Deletions (D Serial Genome Reduction)

Making D-Deletions.

The scarless TREC (tandem repeat coupled with endonuclease cleavage) deletion method was used to generate a series of reduced genomes in yeast. The scarless TREC deletion has been described in Lartigue et al., Science 325:1693-1696 (2009) and Noskov et al., Nucleic Acids Res. 38:2570-2576 (2010) which are hereby incorporated by reference. Six insertion element (IS) and two genes (MMSYN1_460 and _463) flanking one of IS element were sequentially deleted in the genome of JCVI-Syn1.0 Δ1-6 to produce the Syn1/ΔREΔIS genome. The Syn1/ΔREΔIS genome has been described in Karas et al., Nature methods 10:410-412 (2013), which is hereby incorporated by reference. Based on the Tn5 insertion data, twenty-two clusters were selected and subjected to deletion sequentially in the Syn1/ΔREΔIS genome to produce 22 strains (D1 to D22). In each round of the deletion, the genome was tested for viability by transplantation. The detailed information of deleted gene clusters is shown in the Table 9.

TABLE 9 Stepwise D-series deletions of JCVI Syn1.0. number deletion of Δ gene deletion genome no. of genes strains size (bp) genes names coordinates size (bp) deleted JCVI Syn1.0 1,078,809 Syn1.0ΔRE 16,626 17 RE systems 1,062,183 17 Syn1.0ΔREΔIS 13,553 14 IS elements 1,048,690 31 D1 69,607 41 0550-0591 657179 . . . 728756 979,083 72 D2 10,014 6 0698-0703 832678 . . . 842691 969,069 78 D3 24,910 16 0889-0904 1049414-1074323 944,159 94 D4 12,449 7 0180-0186 236820 . . . 249268 931,710 101 D5 8,063 5 0084-0088 112555 . . . 120617 923,647 106 D6 14,716 6 0241-0246 308601 . . . 323316 908,931 112 D7 30,989 27 0734-0770 865972 . . . 907484 877,942 139 D8 11,671 10 0860-0869 1014116 . . . 1025786 866,271 149 D9 22,006 18 0170-0179 222395 . . . 256849 844,265 167 and 0187- 0194 D10 15,364 8 0841-0850  987122 . . . 1003970 828,901 175 D11 12,094 13 0455-0474 563782 . . . 581943 816,807 188 D12 11,301 7 0337-0343 424400 . . . 435700 805,506 195 D13 10,840 5 0272-0276 345463 . . . 356302 794,666 200 D14 9,904 7 0318-0324 402993 . . . 412896 784,762 207 D15 9,631 9 0204-0212 266238 . . . 275868 775,131 216 D16 11,136 8 0118-0125 145646 . . . 156781 763,995 224 D17 6,994 6 0711-0716 849525 . . . 856518 757,001 230 D18 7,481 5 0309-0313 392042 . . . 399522 749,520 235 D19 6,496 5 0854-0857 1005325 . . . 1011820 743,024 240 and 0858 D20 5,516 4 0673-0676 806351 . . . 811866 737,508 244 D21 9,443 5 0594-0598 731220 . . . 740662 728,065 249 D22 6,205 6 0019-0024 36687 . . . 42891 721,860 255 * the gene annotation and sequence coordinate are based on JCVI-Syn1.0 a: MMSYN1_0921-0922; _0449-0450; _0460-0463; _0735-0736; _0780-0781; _ 0265 + _0267 b: deletion of 6 regions: IS1 (35184 . . . 36668), IS3 (553595 . . . 555079), IS4+ (566641 . . . 572708), IS5 (867317 . . . 868801), IS6 (917601 . . . 919085), and IS8 (1000877 . . . 1002361)

The Scarless TREC Deletion Method for Producing the D-Series Deletions.

The TREC method was used to produce scarless deletions (FIG. 23). The design of a knock-out cassette is described in the Experimental Materials and Methods section, except the length of a repeated sequence was reduced to 50 bp, illustrated in FIG. 23. Unique knock-out cassettes were produced by 2 rounds of PCR using the Advantage HD Polymerase (Clontech) according to the manufacturer's instructions. The first round of PCR was performed for 18 cycles using the pCORE3 plasmid as a DNA template and a primer pair 1. The second round of PCR was performed for 22 cycles using the first round PCR product as DNA template and primer pair 2. Chimeric primers in the first round PCR would generate a CORE cassette flanked by a 50 bp repeated sequence on the 5′ end and 50 bp sequences for homologous recombination on the 3′ end. The generation of a CORE cassette using chimeric primers in the first round of PCR has been described in Noskov et al., Nucleic Acids Res. 38:2570-2576 (2010), which is hereby incorporated by reference. Chimeric primers in the second round PCR would generate a final knock-out cassette containing, from the 5′ to 3′ end, a 50 bp for homologous recombination, 50 bp repeated sequence, and a 50 bp for homologous recombination, illustrated in FIG. 23. The second round of PCR product was purified by the MinElute PCR Purification Kit (Qiagen). Approximately 0.5 to 1 μg purified PCR product was used for yeast transformation by the lithium acetate method.

The procedure of TREC deletion and cassette recycling was described previously in Fraser et al., Science 270:397-403 (1995) and Fleischmann et al., Science 269:496-512 (1995), which are hereby incorporated by reference. Briefly, after transformation, cells were plated out on SD (−) URA. Clones were screened by PCR analysis for the boundaries between the cassette and target site. Positive clones were grown on the YEPG media to induce the expression of the endonuclease I-SceI. A double strand cleavage by the endonuclease promoted a homologous recombination between two repeated sequences, leading to removal of the CORE cassette. After induction, cells were grown in SD (−) HIS (+) 5-FOA to select for the removal of the cassette. The precise recycling of the cassette was verified by junction PCR (FIG. 23). Primers were designed for screening knock-out and CORE3 cassette recycling. Primers were designed to amplify the CORE3 cassette for the deletion of gene clusters, and to detect junctions of the CORE3 cassette insertion and the cassette recycling (pop out).

All together, this example shows how genes in a genome can be classified as essential e-genes, non-essential n-genes, quasi-essential, and growth-impaired i-genes.

Example 3 Retention of Quasi-Essential Genes Yielded Eight Viable Segments

This example shows that the retention of quasi-essential genes yielded eight viable segments, but no complete viable genome.

To improve on the design of the HMG, a reduced genome was redesigned using the Tn5 and deletion data described in the Experimental Materials and Methods section and Example 2. This reduced genome design (RGD1.0) achieved a 50% reduction of Syn1.0 by removing approximately 90% of the n-genes (Table 1). In a few cases n-genes were retained if their biochemical function appeared essential or if they were singlet n-genes separating 2 large e- or i-gene clusters. This approach was employed to increase the possibility that the segments and the assembled genome would be viable. To preserve the expression of genes upstream and downstream of deleted regions the design rules used in the HMG design in Example 1 was followed.

The 8 segments of RGD1.0 were chemically synthesized and each synthetic reduced segment was inserted into a ⅞^(th) JCVI-Syn1.0 background in yeast using recombinase-mediated cassette exchange (RMCE) (Experimental Materials and Methods section, FIG. 10). RMCE has been described in Noskov et al., Biol. Proced. Online. 17, 6 (2015), which is hereby incorporated by reference. Each ⅛ RGD+⅞ Syn1.0 genome was then transplanted out of yeast to test for viability. Each of the 8 reduced segments produced a viable transplant; however, segment 6 gave a very small colony only after 6 days. On further growth over the next 6 days, sectors of faster growing cells developed (FIGS. 11A-11G). Several isolates of the faster growing cells were sequenced and found to have destabilizing mutations in a transcription terminator that had been joined to an essential gene when the non-essential gene preceding it had been deleted (FIGS. 12, 14). Another mutation produced a consensus TATAAT box in front of the essential gene (FIG. 13). This illustrates the potential for expression errors when genes are deleted, but shows that these can sometimes be corrected by subsequent spontaneous mutation. Ultimately, a promoter that had been overlooked and erringly deleted was identified. When this region was resupplied in accordance with the design rules, cells containing the redesigned segment 6 grew rapidly. This solution was incorporated in later designs.

When all eight reduced RGD1.0 segments, including self-corrected segment 6 were combined into a single genome, a viable transplant was not obtained (see the Experimental Materials and Methods section). The eight RGD1.0 segments were mixed with the eight Syn1.0 segments to perform combinatorial assembly of genomes in yeast (see the Experimental Materials and Methods section). A number of completely assembled genomes were obtained in yeast that contained various combinations of RGD1.0 segments and Syn1.0 segments. When transplanted, several of these combinations gave rise to viable cells (Table 4B). One of these (RGD2678), containing RGD1.0 segments 2, 6, 7, and 8 plus Syn1.0 segments 1, 3, 4, and 5 with an acceptable growth rate (105 min doubling time versus 60 min for Syn1.0) was analyzed in more detail.

All together, these data indicate that several combinations of the reduced RGD1.0 segments and Syn1.0 segments gave rise to viable cells even though all eight reduced RGD1.0 segments when combined into a single genome did not give rise to a viable transplant.

Example 4 Discovery of Essential Function Redundancies (EFRs) Contributed to Obtaining a Complete Viable Genome

This example shows that the discovery of essential function redundancies (EFRs) contributed to obtaining a complete viable genome.

It was suspected that the failure of the RDG1.0 design to yield a complete viable genome was because of undiscovered essential function redundancies (EFRs) carried by more than one segment. In bacteria, it is common for certain essential (or quasi-essential) functions to be provided by more than one gene. The genes may or may not be paralogs, and in fact, often are not. Suppose gene A and gene B, each supply the essential function E1. The pair represents an EFR. Either gene can be deleted without loss of E1, so each gene by itself in a single knockout study is classified as non-essential. However, if both are deleted, the cell will be dead because E1 is no longer provided. EFRs are common in bacterial genomes, although less so in genomes that have undergone extensive evolutionary reduction such as the mycoplasmas. And thus, undiscovered EFRs in which gene A had been deleted from one segment and gene B from another segment can facilitate the generation of a viable genome with a reduced size. Each RDG1.0 segment was viable in the context of a ⅞ Syn1.0 background, but when combined the resulting cell was non-viable, or grew more slowly in the case of a shared quasi-essential function. The number of redundant essential functions present in different segments were not known, but at least for segments 2, 6, 7, and 8 none of the genes with shared essential functions was deleted and therefore when combined these four segments gave a viable cell.

To discover these EFRs, RGD2678 obtained in Example 3 was subject to Tn5 mutagenesis and it was found that some n-genes in the Syn1.0 segments 1, 3, 4, and 5 had converted to i- or e-genes in the genetic context of RGD2678 (Table 2). Without being limiting to a particular theory, it is believe that these genes encoded EFRs of which one member of the redundant pair had been deleted in RGD2678.

In addition, 39 gene clusters and single genes that had been deleted in the design of RGD1.0 segments 1, 3, 4 and 5 were examined (Table 5). These were deleted one at a time in an RGD2678 background (Tables 5, 6) and tested for viability by transplantation. No transplants, or slow growth, were obtained in several cases suggesting they contained one or more genes functionally redundant with genes that had been deleted in segments 2, 6, 7, or 8.

The combined Tn5 and deletion data identified 26 genes (Tables 2, 10) as candidates for adding back to RGD1.0 segments 1, 3, 4 and 5 to produce a new RGD2.0 design for these segments (Tables 1, 2, FIG. 7). Table 10 shows the 26 genes for the redesign of RGD segments 1, 3, 4, and 5. 4 RGD1.0 version of segments 1, 3, 4, and 5 were re-synthesized by adding back 26 genes to produce RGD2.0-1, -3, -4, and 5.

TABLE 10 26 genes for the redesign of RGD segments 1, 3, 4, and 5. Segment systematic name gene product 1 MMSYN1_0035 conserved hypothetical protein MMSYN1_0036 D-lactate dehydrogenase MMSYN1_0037 malate permease MMSYN1_0038 conserved hypothetical protein MMSYN1_0051 conserved hypothetical protein MMSYN1_0054 AhpC/TSA family protein MMSYN1_0060 putative membrane protein MMSYN1_0077 putative hydrolase of the HAD family MMSYN1_0078 putative hydrolase from alpha/beta family MMSYN1_0080 conserved hypothetical protein 2 MMSYN1_0217 glycerol uptake facilitator protein MMSYN1_0218 glycerol kinase MMSYN1_0219 glycerol oxydase MMSYN1_0232 pantetheine-phosphate adenylyltransferase MMSYN1_0245 putative membrane protein MMSYN1_0246 E1-E2 ATPase subfamily, putative MMSYN1_0251 conserved hypothetical protein MMSYN1_0252 oxidoreductase MMSYN1_0256 Amino acid permease superfamily protein MMSYN1_0275 putative lipoprotein 3 MMSYN1_0332 conserved hypothetical protein MMSYN1_0338 putative lipoprotein 4 MMSYN1_0444 endopeptidase O MMSYN1_0477 conserved hypothetical protein MMSYN1_0494 putative N-acetylmannosamine-6- phosphase 2-ipimeras MMSYN1_0504 conserved hypothetical protein

An assembly was carried out in yeast using the newly designed and synthesized RGD2.0 segments 1, 3, 4, and 5 together with RGD1.0 segments 2, 6, 7, and 8 (Tables 7, 4B). This assembly was still not viable, but substituting Syn1.0 segment 5 for RGD2.0 segment 5 resulted in a viable transplant. Working with this strain, a cluster of genes (0454-0474) was deleted from the Syn1.0 segment 5 and replaced another cluster of genes (0483-0492) with gene MMSYN1_0154 (FIGS. 8, 24, Table 11).

Gene MMSYN1_0154 was originally deleted from segment 2 in the RGD1.0 design but was re-classified as quasi-essential in the RGD2678 background. The described revision of Syn1.0 segment 5 in the RGD2.0 genetic context gave a viable cell, which was referred to as JCVI-Syn2.0 (abbreviated Syn2.0, see FIG. 25). With Syn2.0, it was achieved for the first time, a minimized cell with a genome smaller than that of the smallest known natural bacterium M. genitalium. Syn2.0 doubled in laboratory culture every 92 minutes, and its genome was 576 kb in size and contained 478 protein and 38 RNA coding genes.

Detailed Derivation of the JCVI-Syn2.0 Genome from 7RGDs+WT5 Genome

Among all M. mycoides strains with various intermediate RGD constructs, clone 48 was the smallest genome with an acceptable growth rate (<120 min). Step-wise cluster deletions were used to explore a maximum reduction within the WT 5 segment in the clone 48. Of 39 genes and gene clusters (Table 5), 3 clusters (33, 36, and 37) located in WT 5 segment (Table 5) were selected for deletion by marker replacement. A number of deletion constructs were generated and transplanted. These constructs included all single cluster deletions, a double cluster deletion (Δ33Δ36), and a triple cluster deletion (Δ33Δ36Δ37). All constructs were able to produce viable transplants. In addition, the genome with a deletion region, called cluster 0483-0492, covering the cluster 36, 37, and two genes (MMSYN1_0487 and 0488) between these 2 clusters was also able to produce viable transplants. At this point, the size of the genome after the triple cluster deletion had been reduced to ˜564 kb which was smaller than the 580 kb genome of M. genitalium which was the smallest organism that had been cultured in the laboratory. The growth rate of the triple cluster deletion was about 120 min which was about 2 times slower than that of Syn1.0.

M. mycoides contained two copies of the leucyl aminopeptidase gene (MMSYN1_0154 and 0190). These two genes were originally categorized as n-genes, thus were deleted in the RGD1.0 design. However, data from the Tn5 mutagenesis on the D10 genome indicated that lack of both genes impaired cell growth, thus adding back a leucyl aminopeptidase gene to clone 48 or its reduced derivatives should facilitate cell growth.

A complementation was carried out by replacing the cluster 0483-0492 with gene 0154 in a clone 48 lacking the cluster 33 (Table 10). A CORE6 cassette used to delete the cluster 33 was first recycled in the genome with double cluster deletion (Δ33Δ36), followed by insertion of the gene 0154 to the genome and cassette recycling (FIG. 24). After genome transplantation, multiple clones were isolated and characterized. Once the leucyl aminopeptidase gene was inserted into the genome, transplanted cells exhibited an improved growth rate. The final 576 kb JCVI-Syn2.0 genome was confirmed by sequencing.

FIG. 24 is a schematic illustration of genome engineering to produce the Syn2.0 in yeast. A double clusters deletion (33 and 36) in the 7RGD+WT5 genome was produced by 2 rounds of deletions by the CORE6 cassette containing the K1URA3 and the TRP1 marker, respectively. Step 1, the CORE6 cassette was seamlessly recycled via homologous recombination between 2 repeated sequences flanking the cassette. Step 2, the CORE6 cassette was then used again to replace the region covering both cluster 33 and 36, and two genes (MMSYN1_0483 and 0492). Step 3, MMSYN1_0154 was inserted into the genome via a knock-in module, followed by the cassette recycling.

Table 11 shows the genes that had been deleted in the design of RGD2.0-5. The final Syn2.0 genome was produced by deletion of the cluster 33 (0454-0474) and replacement of the cluster 0483-0492 with MMSYN1_0154 in clone 48. See Table 9 for segment composition of clone 48.

TABLE 11 Genes deleted in the design of RGD2.0-5. MMSYN1_0417 cdse MMSYN1_0436 uracil-DNA glycosylase (UDG) MMSYN1_0454 hypothetical protein MMSYN1_0455 putative membrane protein MMSYN1_0924 conserved domain protein MMSYN1_0460 bacterial surface protein26-residuerepeatprotein MMSYN1_0463 NADH dependent flavin oxidoreductase MMSYN1_0464 lipoate-protein ligase MMSYN1_0465 conserved hypothetical protein MMSYN1_0466 glycine cleavage system H protein MMSYN1_0467 triacylglycerol lipase MMSYN1_0468 lipase-esterase MMSYN1_0469 lipase-esterase MMSYN1_0470 conserved hypothetical protein MMSYN1_0471 hypothetical protein MMSYN1_0472 putative liporotein MMSYN1_0473 ABC transporter, ATP binding protein MMSYN1_0474 ABC transporter, ATP binding protein MMSYN1_0476 N-acetylglucosamine-6-phosphate deacetylase MMSYN1_0480 conserved hypothetical protein MMSYN1_0483 holliday junction DNA helicase RuvA MMSYN1_0484 holliday junction ATP-dependent DNA helicaseRuvB MMSYN1_0485 dihydrolipoamide dehydrogenase MMSYN1_0486 conserved hypothetical protein MMSYN1_0487 conserved hypothetical protein MMSYN1_0488 ribosome biogenesis GTPase YqeH MMSYN1_0489 DNA polymerase IV MMSYN1_0490 papain family cysteine protease, putative MMSYN1_0491 uridine kinase MMSYN1_0492 conserved hypothetical protein MMSYN1_0494 putativeN-acetylmannosamine-6-phosphate2-epimeras MMSYN1_0495 ROK family protein MMSYN1_0496 conserved hypothetical protein MMSYN1_0497 sodium: solute symporter family MMSYN1_0498 N-acetylneuraminatelyase(N-acetylneuraminicacidal MMSYN1_0503 conserved hypothetical protein MMSYN1_0504 rRNA small subunit)S-adenosylmethionine-dependent methyltransferase MMSYN1_0505 putative liporotein Cluster Deletions and Gene MMSYN1-0154 Complementation.

A 2-cluster deletion (Δ33Δ36) in the clone 48 genome was used to create the final RGD genome (JCVISyn2.0). To remove the CORE6 cassette, the recycling construct consisting of the 3′ truncated KanMX4 gene and a 50 bp repeat sequence was produced by 2 rounds of PCR amplification. The 3′ KanMX4 gene was PCR-amplified for 18 cycles using the pFA6a-kanMX4 as template. The second round of PCR was performed using the first round PCR product as DNA template. After transformation, cells were selected on Geneticin G418 plates as described in the Experimental Materials and Methods section. Correct insertions were screened by junction PCR. The procedure for the removal of the cassette was described in the Experimental Materials and Methods section. The resulting genome was subjected to gene knock-in by TREC-IN method (FIG. 7). The CORE6 cassette was generated by 2 rounds of PCR amplification using pCORE6 as DNA template. After transformation, cells were selected on SD minus URA. A correct integration was verified by junction PCR using 2 primer sets at the L junction, and the R junction. Positive clones were subjected to the second round of transformation to insert the gene 0154. The strategy of gene insertion was same as the insertion of gene cluster 0217-0219 as described in the Experimental Materials and Methods section. The 3′ KanMX4 gene was PCR-amplified in 2 rounds and the gene 0154 was PCR-amplified using the Syn1.0 genome as DNA template. The 2 PCR products were purified and co-transformed into yeast and selected on G418 plates. A correct insertion was screened by junction PCR. Positive clones were subjected to the cassette recycling procedure as described in the Experimental Materials and Methods section. A precise removal of the cassette was verified by junction PCR. Multiple positive clones were isolated and subjected to transplantation.

All together, these data indicate that with Syn2.0, a minimized cell with a genome smaller than that of the smallest known natural bacterium M. genitalium was achieved for the first time.

Example 5 A Third Design Stage, RGD3.0, with Removal of 42 Additional Genes, Yielded an Approximately Minimal Cell, Syn3.0

This example demonstrates that a third design stage, RGD3.0, with removal of 42 additional genes from Syn2.0, yielded an approximately minimal cell, Syn3.0.

A new round of Tn5 mutagenesis was performed on Syn2.0. In this new genetic background, transition of some i-genes to apparent n-genes was a possibility. The composition of the P4 serial passage population was depleted of original n-genes and the faster growing i-gene knockouts predominated and were called n-genes by the classification rules. Ninety genes were classified as apparently non-essential. These were sub-divided into 3 groups. The first group contained 26 genes frequently classified as i- or e-genes in previous rounds of mutagenesis. The second group contained 27 genes that were classified as i- or borderline i-genes in some of the previous Tn5 studies. The third group contained 37 genes that had previously been classified as non-essential in several iterations of Tn5 mutagenesis involving various genome contexts. To create the new RGD3.0 design these 37 were selected for deletion from Syn2.0 along with two vector sequences, bla and lacZ, and the rRNA operon in segment 6 (Table 12, FIG. 25).

TABLE 12 Non-essential genes deleted from Syn2.0 to yield Syn3.0. Tn5 mutagenized cells were passaged 6 times to deplete quasi-essential genes (last column). MMSYN1 SGI Annotation syn2_P0 syn2_P1 syn2_P2 syn2_P6 _0013 Mycoides cluster lipoprotein, 168 88 145 93 LppA/P72 family _0028 Cold--shock DNA--binding protein 17 12 12 6 family _0031 Heat shock protein 33, redox 31 13 30 13 regulated chapero _0035 Variable surface protein 115 45 91 48 _0036 D--isomer specific 2--hydroxyacid 98 52 73 62 dehydrogenase _0037 Transporter, auxin efflux carrier 97 59 77 62 (AEC) family pr _0038 ATPase (AAA+ superfamily) 106 58 69 60 _0048 Cytidine and deoxycytidylate 32 16 23 13 deaminase zinc--bi _0062 Macrophage Migration Inhibitory 19 7 21 5 Factor _0078 Alpha/beta hydrolase fold family 56 28 46 22 protein _0096 Praline dipeptidase 17 7 6 7 _0217 Glycerol uptake facilitator protein. 113 57 88 58 _0219 FAD/NAD(P)--binding domain 113 53 60 25 _0258 NAD(P)--binding Rossmann--fold 54 21 40 25 domains _0278 PTS system fructose--specific 218 108 183 133 enzyme iiabc comp _0279 Membrane protein 33 20 25 20 _0284 Lysophospholipase Monoglyceride 30 18 19 15 lipase _0333 Lipoprotein, putative (VlcA) 24 6 28 18 _0334 Lipoprotein, putative (VlcB) 34 13 34 25 _0335 Lipoprotein, putative (VlcC) 37 13 30 17 _0336 Phosphotransferase system PTS, IIA 26 10 16 11 component _0351 Holliday junction resolvase RecU 13 2 11 6 _0355 Lipoprotein, PARCEL family 42 16 36 31 _0370 Single--strand binding family protein 23 14 18 12 _0417 Prophage protein (Ps3) 23 13 24 38 _0436 Uracil--DNA Glycosylase| subunit E 34 14 24 16 _0446 Membrane protein 19 6 6 9 _0476 N--acetylglucosamine--6--phosphate 75 44 57 27 deacetylase _0477 Membrane protein 27 20 23 13 _0480 Conserved predicted protein 90 71 100 135 _0496 8 3 9 4 _0497 Solute: sodium symporter (SSS) 117 57 98 74 family transport _0498 N--acetylneuraminate lyase 70 35 46 30 _0514 Membrane family protein 46 25 24 24 _0677 Membrane protein 38 27 30 26 _0829 Hydrolase, TatD deoxyribonuclease e 18 31 18 family prot 47 _0905 6 2 10 4

The 8 newly designed RGD3.0 segments were synthesized and propagated as yeast plasmids. These plasmids were amplified in vitro by RCA (Experimental Materials and Methods section). All 8 segments were then reassembled in yeast to obtain several versions of the RGD3.0 genome as yeast plasmids (Experimental Materials and Methods section). These assembled RGD3.0 genomes were transplanted out of yeast. Several were viable. One of these, RGD3.0 clone g-19 (Table 13) was selected for detailed analysis and named JCVI-Syn3.0.

FIG. 25 shows the three DBT cycles involved in building Syn3.0. This detailed map shows syn1.0 genes that were deleted or added back in the various cycles going from syn1.0 to syn2.0, and finally to syn3.0 (Compare with FIG. 9). The long white dotted arrows indicate the 8 NotI assembly segments. Light grey arrows represent genes that were retained throughout the process. Genes that are deleted in both syn2.0 and syn3.0 are shown in black. White arrows (slightly offset) represent genes that were added back. The original RGD1.0 design was not viable, but a combination of syn1.0 segments 1, 3, 4, 5 and designed segments 2, 6, 7, 8 produced a viable cell referred to as RGD2678. Addition of the genes shown in white resulted in syn2.0, which has 8 designed segments. Additional deletions (shown in dark grey) produced syn3.0 (531,560 bp, 473 genes).

Table 13 shows that eighth molecule RGD3-1 was synthesized with and without rDNA operon I and eighth molecule RGD3-6 was synthesized with and without rDNA operon II in order to generate three RGD3 genomes: (E) absence of rDNA operon I, (F) presence of rDNA operons I and II, and (G) absence of rDNA operon II. Although full-length RGD3 genomes were assembled in yeast for all three, only (F) and (G) genome versions could generate transplants. One transplant from (G), assigned clone 19-1, was further characterized and later named Syn3.0.

TABLE 13 RGD3 genome constructions in yeast and transplantation results. # Full-length RGD3 constructs Transplantation Genome Construction (out of 48) Results (E) RGD3 Δ rDNA 3 0 out of 3 operon I (F) RGD3 3 3 out of 3 (G) RGD3 Δ rDNA 10 2 out of 10 operon II

A final round of Tn5 mutagenesis was performed on Syn3.0 to determine which genes continue to show Tn5 insertions after serial passaging (P4). Non-essential vector genes and intergenic sequences were the most frequent insertion sites. Cells with insertions in genes originally classified as quasi-essential made up almost the whole population of P4 cells that had insertions in mycoplasma genes. The genes in Syn3.0 were then predominantly essential e-genes, or quasi-essential i-genes by the original Syn1.0 classification. Of these, only the i-genes can tolerate Tn5 insertions without producing lethality. The most highly represented in-, i-, and ie-genes are shown in Tables 3A-3C. In addition, there were a dozen genes originally classified as non-essential that continued to retain that classification (Table 3D, Table 8).

All together, these data indicate that the removal of 42 additional genes from Syn2.0 yielded an approximately minimal cell, Syn3.0.

Table 14 summarizes the generation process leading to syn3.0. Starting with JCVI-syn1.0, four rounds of design (i.e., the HMG, the RGD1.0, RGD2.0, and RGD3.0) were made. The first three rounds of design (i.e., HMG, RGD1.0, and RGD2.0) did not yield complete viable cellular genomes. But in each case, one or more of the 8 segments yielded a viable genome when combined with syn1.0 segments for the remainder of the genome. The composition of several of these intermediate strains is listed in Table 14. RGD3.0, named as JCVI-syn3.0, did yield a viable cell.

Table Error! Reference source not found. 14. Genome designs. Genome Cellular genome segment Cellular design Design composition for key viable genome Growth (1) size (2) strains (3) size (4) (5) — JCVI-syn1.0 (syn1.0) − 1079 kb Td = all 8 syn1.0 segments 60 min HMG 483 kb HMG segment 2 + 7/8 syn1.0 1003 kb slow growing RGD1.0 544 kb RGD1.0 segments 2, 6, 7, 8 + 758 kb Td = syn1.0 segments 1, 3, 4, 5 100 min ″ ″ RGD1.0 segments 1, 2, 4, 6, 718 kb slow 8 + syn1.0 segments 3, 5, 7 growing RGD2.0 575 kb RGD2.0 segments 1, 2, 3, 4, 617 kb ? 6, 7, 8 + syn1.0 segment 5 ″ ″ JCVI-syn2.0 (syn2.0) = RGD2.0 576 kb Td = segments 1, 2, 3, 4, 6, 7, 8 + 92 min syn1.0 segment 5 with genes MMSYN1_0454-0474 and MMSYN1_0483-0492 deleted RGD3.0 531 kb JCVI-syn3.0 (syn3.0, all 8 531 kb Td = segments of RGD3.0) 180 min Column (1) lists the four rounds of genome design, or “—” for the starting genome, syn1.0; Column (2) shows the size of the designed genome Column (3) shows the genome composition for viable cell strains. For non-viable designs, a viable strain with the highest number of segments from the design is shown, as well as a more robust alternative for RGD1.0, and a smaller derivative for RGD2.0, named syn2.0); Column (4) shows the size of the corresponding genome in column 3 Column (5) shows a quantitative or qualitative estimate of the growth rate of cells with the genome described

Example 6 Classification of Genes Retained in Syn3.0

This example demonstrates that Syn3.0 retained essential genes for known core cellular functions, but 150 genes cannot be assigned a specific biological function and 80 of these cannot be assigned to a functional category.

Syn3.0 had 442 protein and 35 RNA coding genes. The 477 genes were assigned to five classes: equivalog, probable, putative, generic, and unknown based on the confidence levels of their precise functions (FIG. 26 and Table 8). Many of the genes had been studied exhaustively and their primary biological functions were known.

FIG. 26 shows a BLAST map of proteins in Syn3.0 and homologs found in other organisms. A BLASTp score of 1e⁻⁵ was used as the similarity cutoff. Functional classifications (equivalog [233 genes]; probable [58 genes]; putative [36 genes]; generic [83 genes]; and unknown [67 genes]) proceed left to right from nearly complete certainty about a gene's activity (equivalog) to no functional information (unknown). White space indicates no homologs to Syn3.0 in that organism.

The TIGRfam ‘equivalog’ family of HMMs was used to annotate such genes (Haft et al., Nucleic Acids Ress 31:371-373 (2003), ˜49% of the genes). The less certain classes were produced in a stepwise manner. Biological functions could not be assigned to about 31% of the genes in the generic and unknown classes. Nevertheless, potential homologs for a number of these were found in diverse organisms. Many of these genes may represent universal proteins whose functions were yet to be characterized. Each of the five sectors had homologs in species ranging from mycoplasma to man. However, some of each annotation class is blank, indicating that no homologs for these genes were found among the 15 organisms chosen for display. Since mycoplasmas evolve rapidly, some of the whitespace in FIG. 26 corresponds to sequences that have diverged so far from the norm as to align poorly with representatives from other organisms.

Table 15 shows the assignment of Syn1.0 genes to 30 functional categories and indicates how many were kept or deleted in Syn3.0. Of the 424 deleted genes, the largest group was the unassigned genes; 133 out of 213 were deleted. All of the 73 mobile element and DNA modification and restriction genes were removed, as well as most genes encoding lipoproteins (71 out of 87). Just these 3 categories alone accounted for 65% of the deleted genes. In addition, because of the rich growth medium used in the examples supplied almost all needed small molecules, many genes involved in transport, catabolism, proteolysis, and other metabolic processes had become dispensable. For example, because glucose was plentiful in the medium, most genes for transport and catabolism of other carbon sources were deleted (32 out of 36), while all 15 genes involved in glucose transport and catabolism were retained.

TABLE 15 JCVI-Syn1.0 genes listed by functional category and whether kept or deleted in JCVI-Syn3.0. Categories in bold type were mostly kept in Syn3.0 while those in non-bold type were depleted in Syn3.0. Functional Category Keep Delete Glucose transport & catabolism 15 0 Ribosome biogenesis 14 1 Protein export 10 0 Transcription 9 0 RNA metabolism 7 0 DNA topology 5 0 Chromosome segregation 3 0 DNA metabolism 3 0 Protein folding 3 0 Translation 89 2 RNA (rRNAs, tRNAs, small RNAs) 35 4 DNA replication 16 2 Lipid salvage and biogenesis 21 4 Cofactor transport and salvage 21 4 rRNA modification 11 4 tRNA modification 14 5 Efflux 7 3 Nucleotide salvage 19 8 DNA repair 8 6 Metabolic processes 11 9 Membrane transport 32 31 Redox homeostasis 4 4 Proteolysis 10 11 Regulation 9 10 Unassigned 80 133 Cell division 1 3 Lipoprotein 16 71 Carbon source transport and catabolism 4 32 Acylglycerol breakdown 0 4 Mobile elements and DNA restriction 0 73 Total 477 424

In contrast, almost all of the genes involved in the machinery for reading and expressing the genetic information in the genome and in assuring the preservation of the genetic information from generation to generation were retained. The first of these two fundamental life processes, expressing the genetic information as proteins, required retention of 201 genes in the categories of transcription, regulation, RNA metabolism, translation, protein folding, protein export, RNA (rRNA, tRNA, small RNAs), ribosome biogenesis, rRNA modification, and tRNA modification. The second of these two fundamental processes, preservation of genome sequence information, required retention of 36 genes in the categories of DNA replication, DNA repair, DNA topology, DNA metabolism, chromosome segregation, and cell division. These 2 processes together required 237 (50%) of the 477 total genes in Syn3.0 (FIG. 27).

FIG. 27 shows the partition of genes into four major functional groups. Syn3.0 had 477 genes. Of these, 80 had no assigned functional category (Table 15). The remainder can be assigned to 4 major functional groups: (1) expression of genome information (201 genes, 42%); (2) preservation of genome information (36 genes); (3) cell membrane structure and function (76 genes, 16%); and (4) cytosolic metabolism (84 genes, 18%). The percentage of genes in each group is indicated.

In addition to the two important biological processes described above—that is, the process of expressing genetic information and the process of preservation of genome sequence information), another major component of living cells is the cell membrane that separates the outer medium from the cytoplasm and governs molecular traffic into and out of the cell. It is an isolatable structure and many of the Syn3.0 genes coded for its protein constituents. Since the minimal cell was largely lacking in biosynthesis of amino acids, lipids, nucleotides, and vitamins, it depended on the rich medium to supply almost all these required small molecules. This necessitated numerous transport systems within the membrane. In addition, the membrane was rich in lipoproteins. Membrane related genes accounted for 76 (16%) of the 477 total Syn3.0 genes. Included categories from Table 15 are lipoproteins, cofactor transport, efflux systems, and other membrane transport systems. Finally, 84 (18%) genes primarily involved in cytosolic metabolism were retained in the categories of nucleotide salvage, lipid salvage and biogenesis, proteolysis, metabolic processes, redox homeostasis, carbon source transport and catabolism, and glucose transport and catabolism (FIG. 27).

Without being limited by a particular theory, it is believe that most of the 80 genes not assigned to a functional category belonged to one or another of these same 4 major groups. Among these 80 genes, 67 had completely unknown function and 13 had generic assignments, for example a hydrolase for which neither the substrate nor the biological role was discernable. The other 70 of the 83 genes in the generic class were assigned to a functional category on the basis of their generic assignment. For example, an ABC transporter was assigned to membrane transport even though the substrate was unknown. Some of these unassigned essential genes matched domains of unknown function (“duf”s) that had been found in a wide variety of organisms.

Example 7 Characterization of Syn3.0

In this example, the growth characteristics of Syn3.0 were studied.

Growth Rate of Syn3.0

Comparison of Syn3.0 to the starting cell Syn1.0 (FIG. 28A) showed that both had a similar colony morphology, characteristic of the natural, wall-less M. mycoides subsp. capri on which the synthetic Syn1.0 genome was originally based. Syn1.0 has been described in Gibson et al., Science 329:52-6 (2010), which is hereby incorporated by reference. The smaller colony size of Syn3.0 suggested a slower growth rate and possibly altered colony architecture on solid medium. A corresponding reduction in the growth rate of Syn3.0 in static liquid culture (FIG. 28B), from a doubling time of ˜60 minutes (min) for Syn1.0 to ˜180 min, confirmed the lower intrinsic rate of propagation for Syn3.0. This rate, however, greatly exceeded the 16 hour (h) doubling time of M. genitalium, described in Jensen et al., J. Clin. Microbiol. 34:286-91 (1996).

In contrast to the reduction in growth rate, striking changes in macro- and microscopic growth properties of Syn3.0 cells were found. Whereas Syn1.0 grew in static culture as non-adherent planktonic suspensions of predominantly single cells with a diameter of ˜400 nm, Syn3.0 cells under the same conditions formed matted sediments. The growth of Syn1.0 in static culture has been described in Gibson et al., Science 329:52-6 (2010). Microscopic images of these undisturbed cells revealed extensive networks of long, segmented filamentous structures along with large vesicular bodies (FIG. 28C), particularly prevalent at late stages of growth. Both of these structures were easily disrupted by physical agitation, yet such suspensions contained small replicative forms that passed 0.2 μm filters to render colony forming units (CFU). This same procedure retained 99.9% of the CFU in planktonic Syn1.0 cultures.

FIGS. 28A-28D show the comparison of Syn1.0 and Syn3.0 growth features. The two panels of FIG. 28A compare colony sizes and morphologies of Syn1.0 and Syn3.0 cells derived from 0.2 μm-filtered liquid cultures diluted and plated on agar medium for 96 h (scale bars=1.0 mm). FIG. 28B shows that the growth rates in liquid static culture determined using a fluorescent measure (RFU) of dsDNA accumulation over time to calculate doubling times (td). FIG. 28C shows native cell morphology in liquid culture imaged in wet mount preparations using differential interference contrast microscopy (scale bars=10 μm). Arrowheads indicate assorted forms of segmented filaments (white) or large vesicles (black). FIG. 28D are scanning electron micrographs of Syn1.0 (left, scale bars=200 nm) and Syn3.0 (middle, scale bars=200 nm and right, scale bars=1 μm). The panel on the right shows a variety of the structures observed in Syn3.0 cultures.

Growth Conditions and Colony Purification

To characterize growth properties of JCVI-Syn1.0 and derivative transplant strains with reduced genomes, cultures were grown at 37° C. in SP-4 liquid medium (containing 17% fetal bovine serum) or on solid medium of the same composition, supplemented with 1% agarose. Initial transplant colonies obtained under selection were picked and propagated in SP4 liquid medium without selection. Static liquid cultures were mixed by trituration and passed through 0.2-μm syringe filters (Acrodisc®, Pall Life Sciences) with gentle pressure. The filtrate was immediately diluted in SP4 medium and 10-fold dilutions were plated on solid SP4 agarose medium. Well-separated colonies from near-limit dilutions were imaged for comparison of size with a stereomicroscope (SZM-45T2, AmScope) and picked for subsequent growth and molecular genetic or phenotypic characterization. Notably, all populations analyzed were filter cloned by this procedure and ultimately were propagated from replicative units that passed through 0.2-μm filters.

Measurement of Growth Rates

To avoid factors that can confound both the measurement of mycoplasma cell growth and the comparison of cells with altered genome content (e.g. differences in the mode of replication, physical aggregation, rates of cell death, altered metabolic indicators, or interference by serum proteins in growth medium), a method was developed (PMID: 25654978, PMID: 25101070) to compare replication rates by a direct measure of cell-associated nucleic acid. Specifically the fluorescent stain Quant-iT™ PicoGreen® (Molecular Probes®, Invitrogen™) which binds dsDNA (and to a far lesser extent dsRNA) was used to quantify the rate of increase during logarithmic-phase cultures in liquid medium.

Procedure To measure logarithmic growth rates, mycoplasma transplants were grown in static, planktonic culture at 37° C. in SP4 liquid medium (without tetracycline or Xgal). Overnight late-logarithmic phase cultures were diluted approximately 500-fold with pre-warmed medium and distributed in replicate 0.80-mL aliquots into graduated 1.7-mL microcentrifuge tubes. Individual tubes were removed at selected times and placed on wet ice to arrest growth. To obtain cells without material loss or contaminating medium components, the collected culture aliquots (or controls containing only medium) were underlain in situ with 0.40-mL sucrose cushions (0.5 M sucrose, 20 mM Tris HCl; pH 7.5) and cells were sedimented by centrifugation at 16,000×g for 10 min. The top layer of medium and cushion were removed by vacuum aspiration and the remaining clear cushion was further adjusted to 100-μL without disrupting pellets. Cells were lysed by adding 50 μL of 0.3% (w/v) SDS in TE, pH7.5 (final concentration 0.1%) followed by trituration and incubation at 37° C. for 5 min. Lysates were diluted to 0.01% SDS by adding 1.35 mL of TE, and mixed by rotary inversion for 1 hr at room temperature. To quantify nucleic acid, equal volumes (80 μL) of diluted lysate and Quant-iT™ PicoGreen® reagent (prepared as described by the manufacturer) were mixed in wells of opaque black 96-well plates (Costar, cat. 3915) and incubated in the dark at room temperature for 5 min. Fluorescence was measured using a FlexStation 3 fluorimeter (Molecular Devices) with excitation at 488 nm, emission collected at 525 nm, and a cutoff setting of 515 nm. The net relative fluorescence units (RFU) of samples (after subtraction of RFU from medium control lacking cells), were plotted as log₂ (RFU) vs. time (min) and the doubling times were calculated from the slopes of exponential regression curves (R², FIG. 29) using the formula: doubling time=ln 2/exponential rate.

FIG. 29 shows the correlation of PicoGreen fluorescence with cell concentration. RFU measurements were obtained from a late logarithmic phase culture of JCVI-Syn1.0 cells diluted with SP4 medium in a 2-fold series (right to left) prior to processing. Medium controls generated a value, RFU=14, that was subtracted from each sample to give the net RFU values shown.

Assay Parameters

Exponential curves generated from cultures diluted 2-fold with complete medium prior to sample processing demonstrated a high correlation between log₂ RFU and cell concentration, over a RFU range of approximately 64-fold (FIG. 29). Linear regions of semi-logarithmic plots within this range were used to calculate exponential replication rates from growing cultures. The accuracy and reproducibility of the technique (reflected in R² values) allowed the use of single samples. To avoid minor variables such as batch differences among medium preparations and temperature fluctuations, constructs were compared under identical conditions and within a single experiment.

Light Microscopy

To observe natural cell morphologies in static cultures without manipulation, wet mounts in medium were prepared by depositing 3 μL of settled cells, carefully removed by micro pipette tip from round-bottom culture tubes, onto an untreated glass slide and applying a 18×18 mm cover slip. Light microscopy was performed using a Zeiss Axio Imager 1 microscope with a Zeiss plan/apochromatic 63× oil 1.4 objective and differential interference contrast (DIC) optics.

Electron Microscopy

Cells grown in SP4 medium were centrifuged at room temperature for 4 min at 2,000×g to produce a loose pellet. Medium (950 μl) was removed and replaced with 1 ml of fixative. The fixative solution was 2.5% glutaraldehyde, 100 mM sodium cacodylate, 2 mM calcium chloride and 2% sucrose (fixative was added cold and samples were stored at 4° C.). Cells were immobilized on polyethylenimine or poly-D-lysine coated ITO glass coverslips for 2 min and washed in 0.1 M cacodylate buffer with 2 mM calcium chloride and 2% sucrose for 5×2 min on ice. Cells were post fixed in 2% osmium tetroxide with 2% sucrose in 0.1 M cacodylate for 30 min on ice. Cells were rinsed in double distilled water and dehydrated in an ethanol series (20, 50, 70, 100%) for 2 min each on ice. Samples were critical point dried (with CO₂) and sputter-coated with a thin layer of Au/Pd. Samples were imaged with a Zeiss Merlin Fe-SEM at 2.5 key, 83 pA probe current and 2.9 mm working distance (zero tilt) using the in-lens SE detector.

All together, these data indicate that Syn3.0 and Syn1.0 had similar colony morphology and characteristic of the natural, wall-less Mycoplasma mycoides subsp. capri on which the synthetic Syn1.0 genome was originally based.

Example 8 Study of Reorganized Genomes

In this example, gene order was reorganized to study if gene order is a major contribution to cell viability.

To further refine the genome-design rules, prospects for logically organizing genomes as well as recoding them at the nucleotide level were investigated for clarifying whether gene order and gene sequence were major contributors to cell viability. Surprisingly, gene order was not critical. About an eighth of the genome was reconfigured into seven contiguous DNA cassettes that comprised six biological systems—the seventh cassette contained genes whose system-level assignment was somewhat equivocal. Numerous intergenic regions (i.e. promoters and terminators) were reassigned to new genes, transcription units were broken apart, etc. Overall, fine-scale gene arrangement was dramatically altered (FIG. 30).

FIG. 30 shows the reorganization of gene order in segment 2. Genes involved in the same process were grouped together in the design for “modularized segment 2”. At the far left the gene order of Syn1.0 segment 2 is indicated. Genes deleted in Syn3.0 are indicated by white lines. Retained genes are indicated by grey lines matching the functional groups they belong to, which are shown on the right side of the figure. Each line connects the position of the corresponding gene in Syn1.0 with its position in the modularized segment 2. Black lines represent intergenic sequences containing promoters or transcriptional terminators.

The resulting organism grew as well in the laboratory as a natural counterpart. While it seems that the details of genetic organization impinge upon survival in hyper-competitive natural environments, it was concluded that gene order was not fundamental to the cell viability.

All together, these data indicate that gene order was not fundamental to life itself even though the details of genetic organization impinge upon survival in hyper-competitive natural environments.

Example 9 Recoding and rRNA Gene Replacement Provide Additional Examples of Genome Plasticity

This example demonstrates genome plasticity by recoding and rRNA gene replacement.

The DBT cycle for bacterial genomes allowed the assessment of the plasticity of gene content in terms of sequence and functionality. This included testing modified versions of genes that are fundamentally essential for life. To demonstrate this, it was tested whether an altered 16S rRNA gene (rrs), which is essential, could support life (FIG. 31A). The single copy of the Syn3.0 rrs gene was designed and synthesized to include seven single-nucleotide changes corresponding to those contained in the M. capricolum rrs gene. In addition, helix h39 (35 nucleotides) was replaced with that from a phylogenetically-distant E. coli rrs counterpart. This unique 16S gene was successfully incorporated into Syn3.0 without noticeably affecting growth rate, providing a “watermark” to quickly identify this strain.

The underlying codon usage principles in the M. mycoides genome, which has extreme adenine and thymine (AT) content, was tested. M. mycoides uses “TGA” as a codon for the amino acid tryptophan instead of a stop codon, occasionally uses non-standard start codons, and the codon usage is heavily biased towards high-AT content. This uncommon codon usage was modified in a 5-kb region containing three essential genes (era, recO and glyS) to determine its significance. Specifically, this region was modified to include (i) M. mycoides codon adaptation index (CAI) but with unusual start codons recoded and tryptophan encoded by the TGG codon instead of TGA, (ii) E. coli CAI but tryptophan still encoded by TGA, or (iii) E. coli CAI and standard codon usage (TGG encoding tryptophan) (FIG. 31B). Surprisingly, all three versions were found to be functional and resulted in M. mycoides cells without noticeable growth differences. Without being limited to any particular theory, it is believed that large-scale changes in codon usage may need to accompany modifications in the tRNA dosage levels to ensure efficient translation.

FIGS. 31A-31B show the testing of gene content and codon usage principles using the DBT cycle. FIG. 31A is a diagram of the modified rrs gene showing its secondary structure that was successfully incorporated into the Syn3.0 genome carrying M. capricolum mutations and h39 (inset) swapped with that of E. coli. Positions with nucleotide changes are indicated by black arrows and E. coli numbering is used to indicate the position of M. capricolum mutations. FIG. 31B shows that three different codon optimization strategies were used for modifying the sequence of the essential genes era, recO and glyS by using M. mycoides codon adaption index (CAI) or that of E. coli with the codons TGG or TGA encoding tryptophan. GC-content of the wild type and the genes modified using the three strategies are noted. JCat codon adaptation tool was used for this exercise to optimize the three open reading frames (ORFs) with the exception of the overlapping gene fragment.

All together, these data indicate genome plasticity in terms of sequence and functionality.

Example 10 Defragmentation of a Eukaryotic Genome

Eukaryotic yeast Kluyveromyces marxianus (K. marxianus) has a genome about 11 MB in size and divided among 8 chromosomes. This example describes the defragmentation of the genome of K. marxianus.

Generation of Tri-Shuttle Vector

A yeast centromeric plasmid (YCp) that is capable of replicating and segregating in K. marxianus yeast cells was built and tested. The YCp contains a K. marxianus centromere and origin of replication and a selectable marker, as well as S. cerevisiae and E. coli vector elements. It was found that this “tri-shuttle vector”, which is around 10 kb in size, can be reliably transferred between K. marxianus, S. cerevisiae, and E. coli. Two versions of YCp have been generated: one containing the uracil selectable marker and one containing the histidine selectable marker. The respective K. marxianus auxotroph strains (ura3Δ and his3Δ) have been generated to accommodate these vectors. In addition, counter-selection with the uracil system using 5-Fluoroorotic Acid (FOA) has been established. Uracil is useful in selecting for engineering chromosomes and FOA is useful for selecting against the native chromosome.

Furthermore, to aid homologous recombination, the robust NHEJ (non-homologous end-joining) activity of K. marxianus has been removed by deleting the Ku70 locus. It was found that 0-10 bp overlaps do not lead to recombination and only 30-60 bp of homology is required to promote robust sequence-specific homologous recombination, allowing efficient and accurate genetic assembly similar to S. cerevisiae.

Sequencing, Annotation, and Transcriptomic and Proteomic Analysis of K. marxianus Genome

A K. marxianus strain is modified to be ku70Δ ura3Δ his3Δ and sequenced on both the PacBio and MiSeq platforms. The fully polished complete genome sequence will then be annotated using homology-based strategies, where the function of genes is predicted based on the function of genes with similar sequences in other species. RNA-Seq will be carried out at several growth stages in minimal (defined) growth medium containing glucose, and at 40° C. to maintain thermotolerance. These experiments will help to determine transcript boundaries, identify transcripts potentially missed at the genome annotation stage, quantify the transcription level of genes, and contribute to the interpretation of dispensability data and to create a catalog of promoter locations, strengths, and transcript boundaries. Samples from several genetically engineered strains with minimized genomes, harvested at different growth stages, including comparisons to wild-type versions of K. marxianus will be analyzed using a state-of-the-art-shotgun proteomic method. Mass spectrometry data will be searched with the K. marxianus protein sequence database and quantitatively analyzed to assess dynamic proteome changes resulting from specific growth states and genetic/genomic mutations. Together with other 'omics and phenotypic features, this data will allow an assessment of functional consequences of genomic minimization of strains. This data can aid in generating a metabolic network of the yeast. Based on limitations of the scope of work, we may use fixed growth conditions (e.g. 45° C. in minimal medium containing glucose) to compare different K. marxianus minimized-genome strains.

A GenBank file containing the complete genome sequence of K. marxianus with high accuracy is generated, with all genes (and known functions) annotated, and transcription unit boundaries including promoter locations and strengths indicated.

Identification of Dispensable Genes in the K. marxianus Genome

A comparative genomics analysis between numerous K. marxianus strains, Pichia pastoris, S. cerevisiae, Schizosaccharomyces pombe, and other yeast species is conducted. S. cerevisiae database contains information for each gene and whether it is dispensable, indispensable, or lethal when combined with another gene deletion. Similar information is also available for S. pombe. The data from transcriptomics and proteomics studies described above can be used to determine genes that are transcribed and translated. These “omics” analyses can generate a candidate list of dispensable genes that can then be tested (see below). The comparison can provide information as to how accurately we can predict the fitness consequences of deleting single genes in a given species given what we know about other genes.

To generate a candidate list of dispensable genes, perform a genome-wide transposon mutagenesis study is performed in K. marxianus. The Tn5-transposase system (Epicentre) is used followed by DNA sequencing on a MiSeq instrument to identify the genes that are disrupted. A transposon map is generated and a single chromosome is selected for minimization. Genes heavily hit with transposons on that chromosome is further validated for dispensability and potential consequences to growth rate by directly knocking out that gene in vivo. Growth rates for individual knockouts is noted and scored as E (essential), N (non-essential), or I (pseudo-essential, impaired growth). Only E and I genes are included in the chromosome design (below). N genes, superfluous DNA sequence, intergenic sequence, transposable elements, and introns are excluded. The Tn5 transposition protocol is modified such that N genes are enriched in the analysis through competitive growth. The results of these experiments are compared with the predictions above.

In parallel, algorithms are developed for selecting genes and gene boundaries to be retained. Upstream and downstream elements including localization signals, enhancers, promoters, ribosome-binding sites, and terminators, are retained. Important intergenic sequence (e.g., origins of replication and the centromere) are identified and retained. Prior to generating a DNA sequence for a single minimal yeast chromosome, a paralog analysis is preformed to ensure essential functions remain intact, especially within the minimal chromosome built. Known synthetic lethal data available for other yeasts, for example S. cerevisiae, are used.

A comparative analysis of predicted and empirically-determined non-essential genes; and a file containing a designed DNA sequence for one K. marxianus chromosome with minimized gene content are generated.

Generation of a Minimized K. marxianus Genome

Synthetic Genome Design.

For the design, a computational framework is assembled with the goal of defining a hierarchy of functional gene modules and predicting which modules can safely be deleted. The main inputs for the computational framework are 1) networks module definitions based on the protein interaction and genetic interaction maps that have been previously generated for S. cerevisiae and S. pombe; 2) evolutionary reconstruction of the history of K. marxianus genes; and 3) transcriptomics and proteomics data for K. marxianus. The computational framework is trained using machine learning and the resulting predictions are experimentally tested in incremental levels of complexity.

First, the computational framework is evaluated for how well it can predict single gene essentiality in K. marxianus. The single- and double-deletion data from other yeast species, as well as the paralogy relationships between K. marxianus gene pairs and the transcriptomics and proteomics data are integrated into an algorithm predicting which genes would be expected to have E, I or N phenotype. The predictions are compared with the results of the transposon experiment and the direct knockout experiments in which the entire ORF is replaced with a selectable marker. The precision is quantified and recall at which the computational framework can predict single gene essentiality.

Second, the computational framework is evaluated for how well it can predict the fitness of the deletion of an entire module. The optimization problem to solve by the algorithm, will be to predict which N genes are the least likely to transition to E or I state in a minimized background. To test the validity of our algorithm, a K. marxianus strain with one entire module deleted that is predicted to be dispensable by our computational framework, using, for example, the Green Monster technology (Suzuki et al., Nature Methods, 2011) is empirically constructed. Upon success, the computational framework is used to assign the letters E, N and I to entire gene modules. Only E and I modules are included in the minimal genome design. Genes in N modules, as well as superfluous intergenic sequences, transposable elements and introns with then be excluded from the native chromosome sequence. Important intergenic sequences (e.g., origins of replication and the centromere) are retained.

The chromosome is constructed as four overlapping sections with conserved overlaps inside essential genes. Later, the sections are designed so that all genes belonging to a given module is contiguous. Only exceptions are overlapping genes, which naturally will remain paired, and pleiotropic genes belonging to multiple modules, which will only be represented once in the designed genome. Because it is anticipated that the computational framework will be partially imperfect, reduced and non-reduced quarter molecules are mix and match either combinatorially or in a directed fashion to determine incompatible reduced sections.

Synthetic Chromosome Assembly.

Chromosomes are synthesized by de novo chemically synthesis of oligonucleotides, by amplifying from genomic DNA template, or a combination thereof. For example, each transcription unit in the minimal chromosome design is PCR amplified and includes a unique 40-bp barcoded sequence within overlapping adjacent transcription units (the barcodes overlap and thus direct homologous recombination whether in vitro or in vivo). These PCR products are then individually cloned and sequence-verified. This strategy provides greater flexibility in the subsequent modularization work (see below). The transcription units are assembled (e.g. 400 units→40 cassettes→4 quarters→1 chromosome), using the in vitro and in vivo DNA assembly methods previously established. Briefly, the transcription units are either assembled enzymatically using a one-step isothermal reaction consisting of an exonuclease, polymerase, and ligase, or by co-transformation and assembly in S. cerevisiae cells. In general, transcription units are selected to begin and end 300 bp upstream and downstream of the open reading frame (ORF).

Synthetic Chromosome Installation.

The minimized K. marxianus chromosome is either cloned in S. cerevisiae as a yeast centromeric plasmid or in E. coli as a bacterial artificial chromosome. The chromosome contains a HIS3 marker for selection and maintenance in K. marxianus. K. marxianus donor chromosomes are transferred from either S. cerevisiae or E. coli to K. marxianus by either electroporation or by the spheroplast fusion method. To aid in complete chromosome transplantation, selection is placed on the donor chromosome and counter-selective pressure is placed on the respective recipient chromosome, which is accomplished by using a recipient K. marxianus strain that is a histidine auxotroph and contains the URA3 gene on the respective native chromosome, which can be selected against by growth in the presence of FOA. Bacteria to yeast fusion has been previously demonstrated (Karas et al., Nature Methods, 2013). In this case, bacterial cells are mixed with yeast spheroplasts in the presence of polyethylene glycol and calcium chloride. Similarly, chromosomes from two different species of yeast strains can be combined in the same cell to generate interspecies hybrids. This process also requires the formation of yeast spheroplasts and is promoted by polyethylene glycol (A. Svoboda, Microbiology, 1978).

Certain combinations of gene deletions can be unpredictably lethal, troubleshooting strategies are designed up front. As above, following two stages of assembly, overlapping quarter molecules of the chromosome are constructed. To aid in troubleshooting, non-reduced versions of the quarter molecules (e.g., by TAR cloning or PCR) are constructed, which permits mixing and matching of reduced and non-reduced quarter molecules either combinatorially or in a directed fashion to determine incompatible reduced sections, and ultimately the genes that cannot be simultaneously disrupted. If, for example, only three of the four reduced segments can be simultaneously combined, another round of transposon bombardment on this strain is performed to identify the remaining genes that can be removed in the non-reduced quarter molecule.

A random “add-back” approach is developed. In this approach, a K. marxianus strain that has both the reduced (but incapable of supporting desired level of K. marxianus growth) and non-reduced chromosomes are provided. Plasmid DNA containing a quarter molecule or random sections of the non-reduced chromosome is transformed into these cells. Counter-selection is then applied to remove the complete non-reduced chromosome. If the cells now survive (due to dependence on the plasmid DNA), the plasmid DNA is sequenced to determine the gene(s) that need to be added back to the design. The plasmid DNA can be generated randomly by ligating a sheared population of DNA derived from purified non-reduced chromosomes or in a direct fashion by assembling a single gene or contiguous genes (previously removed in the design) into a plasmid by in vitro DNA assembly. Genome engineering approaches such as CRISPR/Cas9 and TREC (Tandem Repeat Endonuclease Cleavage), which have proven to be useful in S. cerevisiae, is also adapted for K. marxianus to facilitate the add-back of genes.

How to leverage barcodes in the design to distinguish between the native chromosome and the synthetic version is considered. Assuming the non-reduced and reduced chromosome can co-exist in the same cell, it is possible to identify non-expressed E/I genes in the synthetic chromosome by RNA-Seq. Important intergenic elements not incorporated in our design and uncovering mutations are identified.

A living K. marxianus yeast strain containing one minimized chromosome with its non-reduced counterpart eliminated from the cell is produced.

Generation of a Defragmented Version of the Minimized K. marxianus Genome

In parallel to the minimization efforts, chromosome defragmentation is carried out using well-characterized gene sets that are highly likely to be essential. Essential genes and intergenic regions (which will be defined, cloned, and sequence verified above) are used in the defragmentation process. Essential genes are classified according to function (e.g., replication, transcription, translation, metabolism, etc.). All genes and associated regulatory sequences belonging to a given functional module are represented as contiguous DNA. Only exceptions are pleiotropic genes belonging to multiple modules, which are represented once in the designed chromosome.

Assembly of the defragmented chromosome is carried out in a hierarchical fashion, as above. Alternatively, the original barcoded overlapping sequences and link transcription units are retained together, in a specific manner, using ssDNA oligos. In some instances, this latter approach has the advantage of generating fewer errors and can be leveraged to generate combinatorial libraries of chromosomes and sub-assemblies with varying arrangements of transcriptional units. When possible, combinatorial libraries representing thousands of chromosomal variants are assembled and installed in parallel. Survival and ability to form colonies are screened for.

The corresponding non-modularized quarter-chromosome subsections are cloned and sequence-verified to aid in troubleshooting, as discussed above. Prior to complete chromosome defragmentation, quarter molecules are first individually modularized. Once determined to be individually functional, the quarter molecules are further combined until the chromosome is completely defragmented. If a modularized quarter molecule is determined to be non-functional, it is further broken down into smaller modularized parts (e.g., defragmented eighth molecules) to identify the problematic section(s). The add-back and RNA-Seq troubleshooting strategies addressed above are also used.

The following products are generated: (1) a design for a defragmented version of the minimized K. marxianus chromosome generated above; (2) A design-build-test-troubleshoot workflow for constructing minimized and defragmented eukaryotic chromosomes; and (3) data supporting the construction and testing of the minimized K. marxianus chromosome.

Example 11 Mapping Essential and Non-Essential Genes in K. marxianus

This example describes mapping essential and non-essential genes in K. marxianus by non-homologous end joining insertions of a ura3 gene cassette.

The genome of K. marxianus strain Y-6860 G13 Δura3 Ku70+80+ was subjected to insertional mutagenesis using a 1122-bp PCR product carrying the Saccharomyces cerevisiae URA3 gene. Insertion required the Ku70 protein and thus presumably occurred by the non-homologous end joining (NHEJ) pathway. Analysis of eleven independent insertion events showed either precise insertion without loss of genome sequence, or small flanking genomic duplications and deletions. The inserted ScURA3 terminal sequences were unaltered. Large scale mapping of ScURA3 insertions revealed a greater than 2-fold preference per kilobase for intergenic sequence. Some genes contained no insertions (essential), some were sparsely hit, and others were more heavily hit (non-essential). This example shows that insertional mutagenesis can be a potentially useful alternative to transposon mutagenesis in organisms with an active NHEJ pathway.

High frequency insertion of a PCR product of the S. cerevisiae ScURA3 gene into the genome of K. marxianus DMKU3-1042, a thermotolerant yeast strain, by the non-homologous end joining (NHEJ) pathway have been reported (Abdel-Banet et al. Yeast 27, 29 (2010), the content of which is incorporated by reference in its entirety). To show that a high density ScURA3 insertion map analogous to that obtained by global transposon mutagenesis, nine ScURA3 transformants were analyzed by Southern hybridization in Nonklang et al., Applied and environmental microbiology 74, 7514 (2008) (the content of which is incorporated by reference in its entirety). Insertions sites were all different in the nine transformants and one transformant had multiple insertions.

In this example, high frequency URA3 insertional mapping of another strain of K. marxianus (K. marxianus strain G13 ura3Δ Ku70+80+) was generated. Approximately 8×10⁵ ScURA3 transformants were obtained following transformation of a 1122-base linear DNA fragment and selection on uracil-lacking plates. Analysis of the insertions in chromosome 7, the smallest of the 8 chromosomes of K. marxianus showed 98 genes with 2 or more inserts in the central 60% of the gene after six passages of growth. These genes were classified as non-essential.

Preparation of S. cerevisiae URA3 Cassette DNA

A S. cerevisiae URA3 cassette (1122 bp) was PCR-amplified from plasmid pRS316 (ATCC® 77145™) using primers 5′-tgagagtgcaccacgcttttcaattc and 5-cagggtaataactgatataattaaattg. The 5′ OH PCR product was purified using the QlAquick PCR purification kit.

Preparation of Electrocompetent K. marxianus Cells

K. marxianus (G13 ura3Δ Ku70+80+) cells were grown in YPD (Difco™, Becton, Dickinson and Company) at 30° C. to OD600˜1.0. Twenty-four cell culture aliquots (350 μl) were distributed in 50 ml tubes and centrifuged at 3,000 rpm for 5 minutes. Each cell pellet was washed with 50 ml ice cold sterile water, resuspended in 1 ml ice cold water and transferred to a 1.5 ml Eppendorf tube followed by centrifugation at 4600×g for 2 minutes. Each cell pellet was resuspended in 800 μl of LiAC/TE (100 μl 1M Lithium acetate, 100 μl 10×TE, 800 μl water). Twenty microliters of fresh 1 M dithiothreitol was added to each cell suspension followed by incubation at 30° C. for 45 minutes with gentle shaking (˜100 rpm). Cells were washed with 1 ml ice cold sterile water followed by 1 ml ice cold 1M sorbitol. Cells were pooled in 2.4 ml of ice cold 1 M sorbitol.

Electroporations and Serial Passaging of URA3-Transformed Cells

A large-scale preparation of ScURA3 transformant K. marxianus cells was carried out as follows. A total of 23 electroporations were performed. For each electroporation, 353 ng of ScURA3 PCR product DNA was mixed with 100 μl of electrocompetent cells (˜4.4×10⁸ cells). The mixture was transferred into a chilled 2 mm electroporation cuvette and pulsed at 2500 V, 25 μF, and 200Ω. One milliliter of cold YPDS (YPD+1M sorbitol) was immediately added and cells were transferred to a 15 ml culture tube containing 1 ml cold YPDS. Electroporated cells were allowed to recover at 30° C. for 10 h (FIG. 32). The cells (2.1 ml) were then plated on 22.5 cm×22.5 cm CAA-URA plates (2% glucose, 0.6% casamino acid, 25 μg/ml adenine, 50 μg/ml tryptophan, 0.67% YNB (Difco, BD) without amino acids, and 2% agar). Total colonies were estimated by partially counting 2 of the 23 plates (5 of 4 cm×4 cm squares per plate; one in the center, four at the corners). Colonies were collected and pooled from the 23 plates in ˜200 ml CAA-URA medium (passage P0). We estimated that there was a total of about 8.0×10⁵ colonies representing a transformation efficiency of ˜1×10⁵ colonies/μg of ScURA3 DNA.

Cells were allowed to recover in YPDS medium at 30° C. and plated at intervals on CAA-URA agar plates. As shown in FIG. 32, cell viability increased nearly 10-fold in the interval from 8 to 10 hours, and thereafter the cell number increased at about the doubling rate.

For serial passaging, 125 μl of P0 (˜1.3×10¹⁰ cells/ml) was inoculated into 1 liter of CAA-URA medium and incubated at 30° C. for 24 hours (passage P1). P1 cells (0.5 ml) were inoculated into 250 ml CAA-URA medium and grown for 24 hours (passage P2) and so forth for 6 passages. Aliquots of each passage (P0 to P6) were centrifuged and stored at −20° C. as 200 μl pellets in Eppendorf tubes.

DNA Extraction

A frozen cell pellet from each passage (P0 to P6) was thawed on ice. A 100 μl packed volume of cell pellet was resuspended in 200 μl Qiagen P1buffer. 2 μl of Beta-mercaptoethanol (1.4 M) and 5 μl of Zymolase-100T (20 mg/ml) were added, followed by incubation at 37° C. for 1 h. Cells were lysed by addition of 200 μl Qiagen P2 buffer. The lysate was neutralized by addition of 200 μl Qiagen P3 buffer, followed by centrifugation at 16,000×g for 10 minutes. Supernatant was transferred to a clean microcentrifuge tube. DNA was precipitated by adding 600 μl isopropanol, followed by centrifugation at 16,000 g for 10 minutes. The DNA pellet was dissolved in 100 μl Qiagen EB buffer. RNA was digested with 1 μl of RiboShredder (Epicentre) at 37° C. overnight. After phenol-chloroform extraction, DNA was dissolved in 100 μl EB buffer (56-124 ng/μL).

ScURA3 Marker-Specific Sequencing

For identification of ScURA3-genomic junctions, an approach for mapping the location of transposon insertions was used (Yung et al., Journal of bacteriology 197, 3160 (2015), the content of which is hereby incorporated by reference in its entirety). The tagmentation reaction component of the Illumina Nextera XT library preparation method was used to insert Illumina adapter sequences at random locations throughout the genomic DNA (e.g., Adey et al., Genome biology 11, R119 (2010), the content of which is ehreby incorporated by reference in its entirety). The resulting tagged DNA fragments were amplified using the standard barcoded Illumina P7 adapter and a custom primer containing the Illumina P5 adapter, a random nucleotide spacer, and a homology region to the upstream or downstream edge of the ScURA3cassette. The resulting libraries were size selected, pooled and sequenced on the NextSeq 500 at 2*150 read length. Read 1 contained the ScURA3-genomic DNA junctions.

URA3 Insertions into the K. marxianus Genome were Either Precise or Result in Small Deletions or Duplications of Genomic DNA.

In a study to investigate the nature of the ScURA3 insertions, the ScURA3 cassette DNA was transformed into K. marxianus strain G13 (ura3Δ Ku70+80+). Ten ScURA3 transformant colonies were patched and pooled. The DNA was extracted, and sequenced by Illumina Miseq. The results of sequence analysis of the ScURA3 transformants are shown in Table 16. Among the 10 transformants, there were 11 insertion events. In Table 16, the first column indicates the chromosome and nucleotide position of each insertion; column two indicates whether insertion is precise, or is accompanied by a flanking deletion or duplication event; the third column identifies disrupted genes; and the last column indicates corresponding non-essential genes in S. cerevisiae. The analysis showed 11 different ScURA3 insertion locations, thus one clone contained 2 inserts. Insertions were found in chromosomes 2, 4, 5, 6, and 8. Two insertions were precise with no loss of genome sequence. Two had flanking triplet duplications, and 7 had small flanking deletions, ranging from 1 to 31 bases at the site of insertion. Four of the inserts were in K. marxianus genes homologous to non-essential S. cerevisiae genes and 7 inserted in intergenic regions (Table 16). In all 11 cases, there was no loss of terminal sequences from the ScURA3 cassette.

TABLE 16 Sequence analysis of 10 ScURA3 transformants to determine the nature of the insertion event Chromosome: Type of S. cerevisiae coordinate insertion Gene disrupted homolog Chr2: 31221 precise JCVI1EUKG1592474, ATO2 31215 . . . 32045 Chr2: 1075297- 9 bp deletion JCVI1EUKG1592976, SAP1 1075305 1073059 . . . 1075392 Chr4: 731644- ATG JCVI1EUKT1594332, MNR2 731646 duplication 729525 . . . 732428 Chr4: 743043- 17 bp deletion JCVI1EUKG1594339, RCH1/ 743059 743048 . . . 744460 YMR034C Chr5: 531256- CAC intergenic region 531259 duplication Chr5: 1339737 precise intergenic region Chr6: 601126- single G intergenic region 601127 deletion Chr6: 894933- 31 bp deletion intergenic region 894963 Chr8: 34461- 3 bp deletion intergenic region 34463 Chr8: 752421- 3 bp deletion intergenic region 752423 Chr8: 829922- 31 bp deletion intergenic region 829952 Large-Scale ScURA3 Insertion Mapping of the K. marxianus Genome

Approximately 8.0×10⁵ ScURA3 transformants were pooled from 23 large CAA-URA plates (P0). P0 cells were then serially passaged 6 times. DNA samples for each of passages, P0 through P6, were prepared and sequenced using the marker-specific method described in Methods. Two sets of data were obtained. One set was generated using the KB-URA3-Tn5-lib-5′ primer and NexteraXT P7 primer to generate PCR fragments from the 5′-end of ScURA3 into genomic sequence, and the other set used KB-URA3-Tn5-lib-3′ primer and NexteraXT P7 primer to obtain junction sequences at the 3′-ends of ScURA3 insertions. Insertion sites were precisely identified by Burrows-Wheeler Alignment searching for a gapless match of at least 20 nucleotides to the ends of the ScURA3 cassette followed by a 30 nucleotide flanking sequence which was then used to find a gapless match to the K. marxianus reference genome.

The 5′ and 3′ junction datasets were partially redundant. If sampling were complete, then each insertion site would be supported by both 5′ and 3′ junction sequences. However, because small duplications or deletions of genomic sequence at the junctions may occur, and because the junction sequence datasets were incomplete, it is not possible to definitively distinguish between insertion sites that occur within a few bases of each other. As a conservative approach to managing this redundancy, all insertion sites within 10 bp of one another across both data series were counted as a single insertion event. Without being bound by any particular theory, it is believed that this may result in under-counting of insertions in some cases.

Intergenic insertions slightly outnumbered those in genes. However, intergenic space only accounted for approximately 30% of the genome, thus the number of intergenic insertions per kilobase was more than twice as great (Table 17). This was to be expected since cells with insertions in essential genes will be lost from the population. For example, in an extreme case where all genes were essential, then only intergenic insertions will be represented. Individual cells in the P6 population were considered likely to had insertions primarily in non-essential genes.

All unique insertions in chromosome 7 were tabulated and mapped to the smallest K. marxianus chromosome (Table 17). A total of 4129 insertions were found in P0 and 4018 in P6. FIG. 33 shows a map of P0 and P6 insertions in a small section of chromosome 7 with examples of putative essential and non-essential genes.

FIG. 33 is a non-limiting exemplary plot of a Section of the K. marxianus Chromosome 7 ScURA3 insertion map. P0 inserts are shown black boxes and P6 inserts are shown as white boxes. A gene was classified as non-essential (n) if there are at least two P6 inserts in the middle two-thirds of the gene, otherwise it was essential (e). Reading from top to bottom, gene assignments were: ne, e, ne, e, e, e, e, e, e, e, ne, and e.

ScURA3 Cassettes are Inserted into the K. marxianus Genome by the NHEJ Pathway

Since there is generally no homology of ScURA3 with the K. marxianus genome at the points of insertion, and since the Ku70 gene is required, the mechanism of insertion involves the NHEJ pathway can be inferred. The detailed NHEJ mechanism may not fully known, but it was clear that the Ku70/Ku80 proteins bind to the ends of the DNA at double stranded breaks and hold them in proximity until local DNA repair and joining occurs. Several other proteins, XRCC4, XLF, and DNA ligase IV may participate in the repair and joining process (e.g., Brouwer et al., Nature 535, 566 (2016), and Sharma et al. Journal of nucleic acids (2010), the content of each is incorporated by reference in its entirety), but it was not known if these participate in the insertion mechanism which differs somewhat from DSB repair. The data in this example suggest that when the ScURA3 cassette DNA enters the cell and migrates to the nucleus, the two ends were complexed with the Ku70/Ku80 proteins and brought together to form a circle (FIG. 34A). The data further suggest that the 3′OH groups at each end then carry out nucleophilic attacks on phosphate groups in close proximity but on opposite strands in the backbone of the genomic DNA in analogy to, for example, the Tn5 transposition mechanism. Depending on the positions of the two phosphates, the insertion was precise (no loss of genome sequence), or produced small duplications or deletions of genomic DNA (FIG. 34B).

Various outcomes may be possible when ScURA3 DNA is introduced into K. marxianus. The 5′OH ends of the ScURA3 PCR product could be immediately phosphorylated and ligated to produce circular DNA. Alternatively, the cassette ends could be joined by NHEJ. In addition, linear concatamers, and circular concatamers could be generated. These non-replicating forms would presumably be diluted out as the cells divide. Finally, linear ScURA3 DNA (including linear concatamers) could insert into genomic DNA as described in the above paragraph. The frequency of these various possible outcomes may account in part for strain differences in relative efficiency of the insertional mechanism.

FIGS. 34A-34B show a non-limiting exemplary schematic illustration of a proposed NHEJ insertion mechanism to explain the observed types of ScURA3/genome junctions. FIG. 34A shows that Ku70/80 protein complexes bind to ends of ScURA3 DNA and hold the ends in close proximity. The 3′OH group at each end of ScURA3 DNA carries out nucleophilic attacks on P-atoms in the K. marxianus DNA backbone. FIG. 34 shows that depending on the relative positions of the attack on the P-atoms on the two strands of the helix, there may be either no loss of genome sequence or small insertions or deletions were produced. In the case of deletions, the 3′-overhangs would be removed by an exonuclease followed by ligation to restore continuity. Ligation is indicated by A.

ScURA3 Insertion Map

Without being bound by any particular theory, it is believed that the NHEJ pathway produces random ScURA3 insertions. The observed insertions were not randomly distributed, which can be substantiated by multiple reasons. For example, about 8.0×10⁵ initial ScURA3 transformants were not all independent since during recovery from electroporation some cells divided prior to plating. Secondly, the sequencing protocol involved PCR amplification of ScURA3/genome junction DNA, and it was expected that the degree of amplification will vary due to the different sizes and base compositions of the amplicons. Thirdly, transformants with inserts in essential genes did not produce colonies and were lost. In addition, insertions in some genes could result in slower growth and depletion from the population. These factors are believed to contribute to the >4-fold excess of intergenic versus intragenic insertions (See FIG. 33).

Table 17 tabulates for chromosome 7 the numbers of unique insertions observed for each passage from P0 to P6. Since cells in the P1 population were descendants of those in the P0 population, cells in P2 were descendants of P1, and so forth, the numbers in each subsequent passage should be equal to or less than in the preceding passage. However, inserts found by sequencing were slightly higher in the middle passages. Without being bound by any particular theory, it is believed that this was an artifact of the depth of sequencing achieved for each passage.

TABLE 17 Analysis of ura3 insertions in K. marxianus chromosome 7 by passage number Passage number P0 P1 P2 P3 P4 P5 P6 Total Unique Insertions 4129 4481 7101 6089 6274 6659 4018 in Chr 7 Total Unique insertions 1912 2082 3472 2893 3007 3253 1744 in Gene-Space Total Unique Insertions 2217 2399 3629 3196 3267 3406 2274 in Intergenic-Space Percent Insertions 46.31% 46.46% 48.89% 47.51% 47.93% 48.85% 43.40% in Gene-Space Percent Insertions 53.69% 53.54% 51.11% 52.49% 52.07% 51.15% 56.60% in Intergenic-Space

Table 17 shows that every P6 insertion should also be present in the P0 population. However, on inspection of a small portion of the K. marxianus ScURA3 insertion map shown in FIG. 33, some P6 insertions had no corresponding P0 insertion, although many did. Thus, it was evident that not all the insertion sites in the DNA samples were detected by our depth of sequence coverage.

Altogether, the data indicate that the ScURA3 insertion map can be used in identifying essential and non-essential genes in K. marxianus and potentially, other organisms with functional NHEJ mechanisms.

Example 12 Insertional Mutagenesis and Transposon Mutagenesis

This example describes comparing ScURA3 insertional mutagenesis with T5-transposon mutagenesis using PEG-LiAC-mediated transformation.

PEG-LiAc-Mediated Transformation.

PEG-LiAc-Mediated transformation has been described in Abdel-Banat et al. Yeast 27, 29 (2010), the content of which is incorporated herein in its entirety. Kluyveromyces marxianus cells were grown in 30 ml of YPD at 30° C. in a 250 ml flask with shaking (150 rpm) for 24 h. Cells were harvested by centrifugation at 3000 rpm for 5 minutes. Cell pellet was resuspended in 900 μl TFB (40% polyethylene glycol 3350, 100 mM DTT, 0.2 M lithium acetate) and transferred to a 1.5 ml Eppendorf tube. Cells were collected by centrifugation at 3000 rpm for 5 minutes, then resuspended in 600 μl TFB. Fifty microliters of cell suspension was mixed with ˜70 ng purified ScURA3 fragment in a 1.5 ml Eppendorf tube, and incubated at 42° C. for 30 minutes. The mixture was resuspended in 100 μl CAA-URA medium, plated on CAA-URA plate, and incubated at 30° C. for 2-3 days.

TABLE 18 ScURA3 insertional mutagenesis vs. Tn5 transposen mutagenesis in K. marxianus via PEG-LiAC-mediated transformation. ScURA3 Transposome (70 ng) (1 μl) Wildtype (G13) 914 transformants 96 transformants ku70Δ (G64) None detected None detected

TABLE 19 ScURA3 insertional mutagenesis via electroporation. ScURA3 (70 ng) [10-hour recovery, 1/21 volume of electroporation was plated] Wildtype (G13) 612 transformants ku70Δ (G64) None detected

TABLE 20 Tn5 transposen mutagenesis via electroporation Transposome (1 μl) [14 hours recovery, 1/7 volume of electroporation was plated] Wildtype (G13) 1107 transformants ku70Δ (G64)  595 transformants

ScURA3 insertional mutagenesis was compared with T5-transposon mutagenesis using PEG-LiAC-mediated transformation (Table 18). T5-transposon mutagenesis had a lower transformation efficiency in wildtype K. marxianus. No transformant was detected when ScURA3 insertional mutagenesis was performed using Aku70 cells. This suggested that ScURA3 insertional mutagenesis depends on NHEJ pathway. The fact that no T5-transposon mutagenesis transformants was found suggested that PEG-LiAc-mediated transformation may not be ideal for delivering T5-transposome into K. marxianus. Indeed, electroporation had a much higher transformation efficiency (Table 19) and can deliver T5-transposome into K. marxianus. Interestingly, the number of transformants generated from Aku70 was lower than wildtype cells (Table 20), suggesting that some free T5-transpson DNA fragment may be inserted in the genome of K. marxianus, similarly to ScURA3 insertional mutagenesis through the NHEJ pathway.

Altogether, the data presented in this Example indicate that ScURA3 insertional mutagenesis depends on NHEJ pathway, and that in some conditions, it can be advantageous to use ScURA3 insertional mutagenesis for studying gene-essentiality in K. marxianus than the T5-transposon mutagenesis method using PEG-LiAc-mediated transformation.

Example 13 Transfer DNA into Kluyveromyces marxianus by Conjugation

This example describes using conjugation to transfer DNA into K. marxianus with an oriT (origin of transfer)-containing plasmid.

An oriT (origin of transfer)-containing plasmid can be transferred into diatom and S. cerevisiae from E. coli through conjugation (Karas et al., Nature Communications 6, 6925 (2015), and Moriguchi et al., PLoSOne 11, e0148989 (2016), the content of each is incorporated by reference in its entirety). This system was adopted for K. marxianus. First, the oriT sequence was inserted in pCC1BAC-LCyeast_(scHis3)-SYN-KM_CENARS between EcoRI and BamHI restriction sites (FIG. 35). Using this plasmid, a protocol of E. coli to K. marxianus conjugation was established.

Conjugation.

E. coli (plasmid to be transferred and helper plasmid) cells were grown in 5 ml LB+Chloramphenicol+Gentamycin medium at 37° C. overnight and K. marxianus were grown in 5 ml YPAD at 30° C. overnight. Cells were harvested by centrifugation at 3000 rpm for 5 minutes. K. marxianus was resuspended in 200 μl LB or SOC and E. coli was resuspended in 700 μl LB or SOC (Do not vertex E. coli). K. marxianus suspension was plated on a dry LB+Chloramphenicol+Gentamycin plate and air dry. E. coli suspension was added on top of the K. marxianus (making sure that the E. coli suspension covers the entire plate). The plate was incubated at 37° C. overnight, then replica-plated onto a selective plate and incubated at 30° C. for 3 days.

pCC1BAC-LCyeast_(scHis3)-SYN-KM_CENARS_oriT was transferred into K. marxianus in the presence of the helper plasmid (pTA-MOB as described in Strand et al., PloS one 9, e90372 (2014), the content of which is whereby incorporated by reference in its entirety). FIG. 36 shows establishment of E. coli to K. marxianus conjugation. E. coli (EPI300) was transformed with plasmids in the bottom table. Eight K. marxianus conjugation colonies were screened for the presence of pCC1BAC-LCyeast_(scHis3)-SYN-KM_CENARS_oriT. Genomic DNA (FIG. 37, left panel); oriT PCR product (FIG. 37, right panel). Conjugation was also utilized to deliver DNA molecules of up to 100 kb from E. coli to K. marxianus via conjugation (Table 21 and FIG. 38). FIG. 38 is a non-limiting exemplary gel electrophoresis photograph showing that large DNA fragment can be transferred from E. coli to K. marxianus via conjugation. Lane 1. #4-55—clone 1; Lane 2. #4-55—clone 2; Lane 3. #4-55—E. coli; Lane 4. #3; Lane 5. NEB 1 kb ladder. (genomic DNA on TAE gel with SYBR Gold Staining)

TABLE 21 E. coli to K. marxianus conjugation (*conjugations of different 1/10^(th) molecules was performed at different times). 1/10^(th) molecule of minimized K. marxianus Chromosome 7 1 2 3 4 5 9 Size 50883 71081 48341 100454 76643 53037 bp bp bp bp bp bp # colonies* 258 3 65 203 17 40 on selective plate

Altogether, the data demonstrate that conjugation can be used to deliver chromosomal segment up to ˜100 kb from E. coli to K. marxianus.

Example 14 Design-Build-Test Cycle in K. marxianus

This example describes a design-build-text cycle using a Cas9-expressing K. marxianus.

The unique method of hierarchical assembly of chromosome 7 of K. marxianus disclosed herein allowed choosing intermediate sub-assembly molecules for redesigning, and testing of the functionality of the redesigned molecules (strategy outlined in FIG. 39). Specifically, the Stage-II subassembly molecules (˜80 kb) was chosen for redesigning and testing, one at a time. As a first step, the replication elements necessary for maintenance in K. marxianus were introduced into these subassembly molecules. Subsequently, these molecules were transformed into K. marxianus strain

expressing Cas9 protein. After establishing stable maintenance of the 1/12th molecule, the corresponding segment would be deleted from chromosome 7 using CRISPR/Cas9. This resulted in a strain where a part of the genome was solely expressed from an episome, which enabled rapid replacement of this molecule with newly designed 1/12th molecules and verify functionality. Ultimately, the information obtained from each of these 1/12th molecules can be combined to create a redesigned chromosome.

Generating a Cas9-Expressing K. marxianus Strain

In order to streamline the process of testing our design using Cas9, a strain which expressed Cas9 on the chromosome was engineered. For this, the ade1 locus was chosen. Adel gene is involved in the biosynthesis of adenosine monophosphate. When this gene is interrupted, the biosynthesis of adenosine monophosphate is also arrested, which leads to the accumulation of P-ribosylaminoimidazole. This compound, upon oxidation under aerobic growth, turns red in color. Thus, interruption of the ade1 locus leads to the accumulation of yeast cells that are “red” in color.

Cas9 expressed from the plasmid was used to introduce Cas9 into the chromosome of K. marxianus, at the ade1 locus. The accumulation of the “red” pigment, led to the easy identification of the cells that were edited at the ade1 locus. The ade1 gene substitution with Cas9 was verified using genomic DNA isolation and subsequent PCR. The sequence of the Cas9 gene with its expression elements (promoter and terminator) was verified using MiSeq (Illumina, San Diego, Calif.).

Verifying the Utility of the Test Cycle Using a Positive Control

Initially, CRISPR/Cas9 based test cycle on “wild-type” subassembly molecule was tested. Specifically, one episomal Stage-II molecule of ˜80 kb (wild-type) was introduced into K. marxianus and the corresponding segment from the chromosome was deleted using CRISPR/Cas9. Hence, in the final strain, ˜80 kb of the genomic content was solely expressed from the episome. The Stage-II molecule, #6_12 was introduced into K. marxianus constitutively expressing Cas9. Shown in FIG. 40 is the verification of the presence of the Stage-II molecule, #6_12 after it was transformed into K. marxianus. As a comparison, the same molecule extracted from E. coli and S. cerevisiae were resolved in parallel on a 1% agarose gel for 3 hrs at 4.5V/cm and post-stained with SYBR-gold. Once the stable replication of the episomal molecule was established, this K. marxianus strain was transformed with gRNAs to direct Cas9-mediated cleavage of the chromosomal segment corresponding to the #6_12 molecule. Ultramer oligonucleotides were used to generate gRNA transcripts for deleting the chromosomal fragment encoded by Stage-II molecule #6-12 using Cas9. An ultramer oligonucleotide was used as a ssDNA donor during the Cas9-mediated deletion the chromosomal fragment encoded by Stage-II molecule #6-12. The ultramer oligonucleotide was used as a ssDNA donor to patch the two chromosomal arms after Cas9-mediated cleavage.

Once the transformation (with gRNAs and ssDNA) was performed cells were plated on selective media and screened using colony PCR. Primers were designed such that if the Cas9-mediated deletion occured, ˜465 bp single band should be observed; else a 300 bp band and a 400 bp band would be seen (wild-type). These primers were used to probe chromosomal deletion of the DNA encoded by Stage-II molecule #6-12.

96 colonies were screened using the above listed primers to probe chromosomal deletion. Every single colony that was screened carried the deletion (FIG. 41). Six colonies from the transformants that tested positive for chromosomal deletion were subjected to DNA extraction to confirm the presence of the #6_12 episome in these strains (FIG. 42).

The chromosomal deletion of the DNA fragment encoded in the episome #6_12 was further verified using quantitative PCR (qPCR) (FIGS. 43-44, Table 22) and pulse-field gel-electrophoresis (PFGE) (FIG. 45). FIG. 43 shows a non-limiting exemplary qPCR design. Primers for four amplicons were designed such that three amplicons would be from different parts of the segment encompassed by #6_12 (amplicons 1-3) and one outside this segment (amplicon 4). qPCR would help determine the relative copy number of the DNA fragment encoded in #6_12 fragment. In the wild-type strain, amplicons 1-3 should be of the same relative amount in a qPCR as amplicon 4; when the episomal DNA #6_12 was introduced, the cell should carry two copies of the segment encoded in #6_12—one in the chromosome, another in the episome: this would result in twice amount of amplicons 1-3 relative to 4; however after the CRISPR/Cas9 mediated deletion, the copy number of segment #6_12 returns to one since there is only one copy of this DNA fragment—in the episome: this would result in the same relative amount of amplicons 1-3 compared to amplicon 4.

Genomic DNA was extracted from the wild-type strain (WT), strain carrying episomal DNA #6_12 (Episome+chromosome (“chr”)) and strain carrying episomal DNA #6_12 but with the corresponding chromosomal fragment deleted (Episome only). 50 ng of genomic DNA was used in each qPCR reaction using primers. Reactions were performed in triplicates and average Ct values were calculated. ΔCt values were calculated for each of the amplicons 1-3 relative to amplicon 4, for all three strains. The Ct and ΔCt values are listed in Table 22 and plotted in FIG. 44. The qPCR results confirmed that in the strain carrying the episomal DNA #6_12, the corresponding chromosomal fragment was indeed deleted using CRISPR/Cas9. In this strain, ˜80 kb of genomic DNA was encoded solely from an episome.

Pulse-field gel-electrophoresis was used to confirm the CRISPR/Cas9 mediated deletion of the chromosomal ˜80 kb fragment in the strain carrying episomal DNA #6_12. For this, genomic DNA was captured in agarose plugs in the following strains: wild-type strain (WT), strain carrying episomal DNA #6_12 (Episome+chr), and strain carrying episomal DNA #6_12 but with the corresponding chromosomal fragment deleted (Episome only).

Following genomic DNA preparation, chromosomes were resolved using Pulse-field gel-electrophoresis (FIG. 45). In the “episome only” strain, a faster-migrating chromosomal species was observed, hence confirming the ˜80 kb deletion using CRISPR/Cas9 in chromosome 7.

TABLE 22 Average and ΔCt values obtained from the qPCR experiment described in FIG. 43. 50 ng gDNA template Fold-difference Avg Ct ΔCt (normalized to amplicon-4) WT Primer set_1 14.90333 −0.18333 1.135504 Amplicon-1 Primer set_2 15.19667 0.11 0.926588 Amplicon-2 Primer set_3 15.17 0.083333 0.943874 Amplicon-3 Primer set_4 15.08667 0 Amplicon-4 Episome + chr Primer set_1 13.45333 −1.20667 2.308038 Amplicon-1 Primer set_2 13.59333 −1.06667 2.094588 Amplicon-2 Primer set_3 13.50667 −1.15333 2.224272 Amplicon-3 Primer set_4 14.66 0 Amplicon-4 Episome only Primer set_1 14.59333 −0.18 1.132884 Amplicon-1 Primer set_2 14.94333 0.17 0.888843 Amplicon-2 Primer set_3 14.79 0.016667 0.988514 Amplicon-3 Primer set_4 14.77333 0 Amplicon-4

Altogether, the data desmonstrate that a design-build-text cycle using a Cas9-expressing K. marxianus and the information obtained from each of these 1/12th molecules can be combined to create a redesigned chromosome.

Example 15 Utilizing the CRISPR/Cas9 Based Test Cycle to Verify Genomic Design of a Minimized Chromosomal Segment

This example describes testing a CRISPR/Cas9-based method to evaluate the chromosomal minimization design.

A CRISPR/Cas9-based method was tested to evaluate the chromosomal minimization design. For this, we identified and built a minimized Stage-II molecule. Unlike the #6_12 Stage-II molecule that was used to evaluate the CRISPR/Cas9 method, the minimized molecule was not a replica of the corresponding chromosomal segment, instead lacked ˜20 kb of genomic material due to minimization. This episome molecule, #2_37 was introduced into K. marxianus using conjugation (FIG. 46).

Once the stable replication of the episomal molecule was established, this K. marxianus strain was transformed with gRNAs to direct Cas9-mediated cleavage of the chromosomal segment corresponding to the #2_37 molecule. The gRNAs were prepared from ultramer oligonucleotides. Ultramer oligonucleotides used to generate gRNA transcripts for deleting the chromosomal fragment encoded by the minimized Stage-II molecule #2_37. Ultramer oligonucleotide was used as an ssDNA donor to patch the two chromosomal arms after Cas9-mediated cleavage. Ultramer oligonucleotide used as a ssDNA donor during the Cas9-mediated deletion the chromosomal fragment encoded by Stage-II molecule #2_37.

Once the transformation (with gRNAs and ssDNA) was performed cells were plated on selective media and screened using colony PCR. Primers were designed such that if the Cas9-mediated deletion occurred, ˜360 bp single band should be observed; else a 350 bp band and a 450 bp band would be seen (wild-type). Primer sequences used to probe chromosomal deletion of the DNA (˜91 kb) encoded in minimized Stage-II molecule 2_37 (˜71 kb). 48 colonies were screened using the above listed primers to probe chromosomal deletion (FIG. 47). The chromosomal deletion was further verified using qPCR and pulsed-field gel electrophoresis (PFGE) disclosed herein.

Genomic DNA was extracted from the wild-type strain (WT), strain carrying minimized episomal DNA #2_37 (Episome+chr) and strain carrying minimized episomal DNA #2_37 but with the corresponding chromosomal fragment deleted (Episome only). Reactions were performed in triplicates and average Ct values were calculated. ΔCt values were calculated for each of the amplicons 1-2 relative to amplicon 3, for all three strains. The Ct and ΔCt values are listed in Table 23 and plotted in FIG. 49. The qPCR results confirm that in the strain carrying the minimized episomal DNA #2_37, the corresponding wild-type chromosomal fragment was indeed deleted using CRISPR/Cas9. In this strain, ˜91 kb of genomic DNA was deleted and instead a ˜71 kb DNA from an episome is sufficient to support growth, thus verifying the genome minimization design in this subchromosomal fragment.

TABLE 23 Average and ΔCt values obtained from the qPCR experiment described in FIG. 48. Fold-difference Avg Ct ΔCt (normalized to amplicon-4) WT Primer set_1 16.72666667 −0.043333333 1.03049202 Amplicon-1 Primer set_2 16.80667 0.036667 0.974905 Amplicon-2 Primer set_3 16.77 0 1 Amplicon-3 Episome + chr Primer set_1 16.23 −0.9 1.866066 Amplicon-1 Primer set_2 16.24667 −0.88333 1.844632 Amplicon-2 Primer set_3 17.13 0 1 Amplicon-3 Episome only Primer set_1 16.38667 −0.23667 1.178267 Amplicon-1 Primer set_2 16.34333 −0.28 1.214195 Amplicon-2 Primer set_3 16.62333 0 1 Amplicon-3

Pulse-field gel-electrophoresis was used to confirm the CRISPR/Cas9 mediated deletion of the chromosomal ˜91 kb wild-type fragment in the strain carrying minimized episomal DNA #2_37. For this, genomic DNA was captured in agarose plugs in the following strains: wild-type strain (WT), strain carrying minimized episomal DNA #2_37 (Episome+chr), strain carrying the minimized episomal DNA #2_37 but with the corresponding wild-type chromosomal fragment deleted (Episome only).

Following genomic DNA preparation, chromosomes were resolved using Pulse-field gel-electrophoresis (FIG. 50). In the “episome only” strain, a faster-migrating chromosomal species was observed, hence confirming the ˜91 kb deletion using CRISPR/Cas9 in chromosome 7.

This example shows that a CRISPR/Cas9-based method can be used to evaluate the chromosomal minimization design by first identifying and building a minimized Stage-II molecule, introducing this Stage-II molecule into K. marxianus by conjugation, transforming this K. marxianus strain with gRNAs to direct Cas9-mediated cleavage of the chromosomal segment, and confirming the CRISPR/Cas9 mediated deletion using pulse-field gel-electrophoresis.

In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims. 

What is claimed is:
 1. A method for generating a minimal synthetic genome of interest, comprising: (a) providing a first genome known to sustain viability of a prokaryotic cell; wherein the first genome has a size of no more than 12 megabase pair (Mb); (b) designing and providing a second genome comprising a reduced number of genes compared to the first genome, wherein designing the second genome comprises modifying the first genome; (c) dividing each of the first and second genomes into at least three corresponding fragments, (d) combining at least one fragment of the second genome with fragments of the first genome to generate a third genome having all the three corresponding fragments, further comprising grouping genes related to the same biological process in the at least one fragment of the second genome prior to combining; or grouping genes related to the same biological process in the third genome after combining; (e) testing the third genome generated in step (d) for sufficiency to sustain viability of the prokaryotic cell; and (f) identifying the third genome as a minimal synthetic genome of interest if it sustains viability of the prokaryotic cell; otherwise genetically modifying the at least one fragment of the second genome and repeating steps (d)-(f) in one or more iterations until a genome that sustains viability of the prokaryotic cell is obtained in the third genome.
 2. The method of claim 1, wherein the first genome is a Mycoplasma genome.
 3. The method of claim 1, wherein the first genome is a multi-chromosome genome.
 4. The method of claim 1, wherein step (b) further comprises testing the second genome for a set of desired properties selected from the group consisting of: growth rate, ratio of growth rate to genome size, expression level of a gene of interest, ratio of viability to genome size, ratio of viability to expression level of a gene of interest, and ratio of growth rate to expression level of a gene of interest.
 5. The method of claim 1, wherein modifying the first genome is based on the information from literature resources, experimental data, or any combination thereof.
 6. The method of claim 5, wherein the experimental data comprises data related to genes of essential function redundancies (EFR), or data obtained from a mutation study of the first genome, a genome related to the first genome, or any combination thereof.
 7. The method of claim 1, wherein at least one of the at least three fragments is a chromosome of the first or second genome, or a portion of a chromosome of the first or second genome.
 8. The method of claim 1, wherein testing the third genome for sufficiency to sustain viability of a cell comprises introducing the genome into a cell or a cell-like system.
 9. The method of claim 1, wherein modifying at least one fragment of the second genome in step (f) further comprises conducting mutation study of the at least one fragment and modifying the at least one fragment at least partly based on the mutation study.
 10. The method of claim 1, wherein step (c) comprises dividing each of the first and second genomes into between 4 and 20 corresponding fragments.
 11. The method of claim 1, wherein at least one fragment of the second genome is present in an extrachromosomal genetic element.
 12. The method of claim 1, wherein the method generates a plurality of third genomes each having all of the at least three fragments.
 13. The method of claim 1, wherein the combining step comprises chemically synthesizing and assembling the fragments of the first and second genomes to generate the third genome.
 14. The method of claim 1 further comprising grouping the genes related to the same biological process as contiguous modules a) in the at least one fragment of the second genome, or b) on the third genome.
 15. The method of claim 14 wherein the same biological process is selected from the group consisting of: transport and catabolism, ribosome biogenesis, protein export, DNA repair, transcription, translation, nucleotide biosynthesis, metabolism and salvage, glycolysis, proteolysis, membrane transport, rRNA modification, and tRNA modification.
 16. The method of claim 14 wherein step (c) comprises dividing each of the first and second genomes into at least 20 corresponding fragments.
 17. The method of claim 14 wherein the same biological process is selected from the group consisting of: transcription, RNA metabolism, translation, protein folding, protein export, a gene encoding RNA, ribosome biogenesis, rRNA modification, and tRNA modification.
 18. The method of claim 1 wherein the same biological process is selected from the group consisting of: expression of genome information, preservation of genome information, cell membrane structure and function, and cytosolic metabolism. 